Materials and methods for treatment of spinocerebellar ataxia type 1 (sca1) and other spinocerebellar ataxia type 1 protein (atxn1) gene related conditions or disorders

ABSTRACT

The present disclosure provides materials and methods for treating a patient with one or more conditions or disorders associated with ATXN1 whether ex vivo or in vivo. For example, the present disclosure provides materials and methods for treating a patient with Spinocerebellar ataxia type 1 (SCA1). Also provided are materials and methods for editing a ATXN1 gene in a cell by genome editing. The present disclosure also provides materials and methods for altering the contiguous genomic sequence of a ATXN1 gene in a cell. In addition, the present disclosure provides one or more gRNAs for editing a ATXN1 gene. Also provided are therapeutics comprising at least one or more gRNAs for editing a ATXN1 gene. In addition, the present disclosure provides therapeutics for treating patients with a ATXN1 related condition or disorder.

FIELD

The present disclosure provides materials and methods for treating apatient with Spinocerebellar Ataxia Type 1 (SCA1) and otherSpinocerebellar Ataxia Type 1 Protein (ATXN1) gene related conditions ordisorders, both ex vivo and in vivo.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational Application No. PCT/IB2018/051037, filed Feb. 20, 2018,which claims the benefit of U.S. Provisional Application No. 62/461,850filed Feb. 22, 2017, the disclosure of each is hereby incorporated byreference in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted oncompact disc and is hereby incorporated by reference in its entirety.The sequence listing consists of a file named170135PCT-Sequence-Listing.txt, created Feb. 11, 2018, and is109,329,000 bytes in size.

BACKGROUND

Genome engineering refers to the strategies and techniques for thetargeted, specific modification of the genetic information (genome) ofliving organisms. Genome engineering is a very active field of researchbecause of the wide range of possible applications, particularly in theareas of human health. For example, genome engineering can be used toalter (e.g., correct or knock-out) a gene carrying a harmful mutation,or to explore the function of a gene. Early technologies developed toinsert a transgene into a living cell were often limited by the randomnature of the insertion of the new sequence into the genome. Randominsertions into the genome may result in disrupting normal regulation ofneighboring genes leading to severe unwanted effects. Furthermore,random integration technologies offer little reproducibility, as thereis no guarantee that the sequence would be inserted at the same place intwo different cells. Recent genome engineering strategies, such as zincfinger nucleases (ZFNs), transcription activator like effector nucleases(TALENs), homing endonucleases (HEs) and MegaTALs, enable a specificarea of the DNA to be modified, thereby increasing the precision of thealteration compared to early technologies. These newer platforms offer amuch larger degree of reproducibility, but still have their limitations.

Despite efforts from researchers and medical professionals worldwide whohave been trying to address genetic disorders, there still remains acritical need for developing safe and effective treatments for ATXN1related disorders and conditions.

SUMMARY

Provided herein are cellular, ex vivo and in vivo methods for creatingpermanent changes to the genome by deleting and/or correcting thetrinucleotide repeat expansion or replacing one or more nucleotidebases, or one or more exons and/or introns within or near theSpinocerebellar Ataxia Type 1 Protein (ATXN1) gene, or otherwiseintroducing insertions, deletions or mutations of at least onenucleotide within or near the ATXN1 gene or other DNA sequences thatencode regulatory elements of the ATXN1 gene by genome editing. Suchmethods can reduce or eliminate the expression or function of aberrantATXN1 gene products and/or restore the wild-type ATXN1 protein activity,which can be used to treat an ATXN1 related condition or disorder suchas Spinocerebellar ataxia type 1 (SCA1). Also provided herein arecomponents and compositions, and vectors for performing such methods.

Provided herein is a method for editing a ATXN1 gene in a cell by genomeediting. The method can comprise: introducing into the cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the ATXN1 gene. The one or more SSBs or DSBs can result in apermanent deletion in the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the ATXN1 gene, thereby restoring the ATXN1 genefunction.

Also provided herein is a method for editing a ATXN1 gene in a cell bygenome editing. The method can comprise: introducing into the cell oneor more DNA endonucleases to effect one or more SSBs or DSBs within ornear the ATXN1 gene or ATXN1 regulatory elements. The one or more SSBsor DSBs can result in one or more permanent insertion, deletion ormutation of at least one nucleotide within or near the ATXN1 gene and/ora permanent deletion in the expanded trinucleotide repeat, therebyreducing or eliminating the expression or function of aberrant ATXN1gene products.

Also provided herein is an ex vivo method for treating a patient havingan ATXN1 related condition or disorder. The method can comprise: editinga patient specific induced pluripotent stem cell (iPSC) within or near aATXN1 gene or other DNA sequences that encode regulatory elements of theATXN1 gene; differentiating the edited iPSC into a neuron or glial cellof the Central Nervous System (CNS); and implanting the neuron or glialcell of the CNS into the patient.

The editing step can comprise: introducing into the iPSC one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theATXN1 gene that results in a permanent deletion of the expandedtrinucleotide repeat or replacement of one or more nucleotide bases, orone or more exons and/or introns within or near the ATXN1 gene and/or apermanent deletion in the expanded trinucleotide repeat, therebyrestoring the ATXN1 gene function.

The editing step can comprise: introducing into the iPSC one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theATXN1 gene or ATXN1 regulatory elements that results in one or morepermanent insertion, deletion or mutation of at least one nucleotidewithin or near the ATXN1 gene, thereby reducing or eliminating theexpression or function of aberrant ATXN1 gene products.

The method can further comprise: creating the iPSC. The creating stepcan comprise: isolating a somatic cell from the patient; and introducinga set of pluripotency-associated genes into the somatic cell to inducethe somatic cell to become the iPSC. The somatic cell can be afibroblast. The set of pluripotency-associated genes can be one or moreof the genes selected from the group consisting of: OCT4, SOX2, KLF4,Lin28, NANOG and cMYC.

Also provided herein is an ex vivo method for treating a patient havingan ATXN1 related condition or disorder. The method can comprise: editinga mesenchymal stem cell within or near a ATXN1 gene or other DNAsequences that encode regulatory elements of the ATXN1 gene;differentiating the edited mesenchymal stem cell into a neuron or glialcell of the CNS; and implanting the neuron or glial cell of the CNS intothe patient.

The editing step can comprise: introducing into the mesenchymal stemcell one or more DNA endonucleases to effect one or more SSBs or DSBswithin or near the ATXN1 gene that results in a permanent deletion ofthe expanded trinucleotide repeat or replacement of one or morenucleotide bases, or one or more exons and/or introns within or near theATXN1 gene, thereby restoring the ATXN1 gene function.

The editing step can comprise: introducing into the mesenchymal stemcell one or more DNA endonucleases to effect one or more SSBs or DSBswithin or near the ATXN1 gene or ATXN1 regulatory elements that resultsin one or more permanent insertion, deletion or mutation of at least onenucleotide within or near the ATXN1 gene and/or a permanent deletion inthe expanded trinucleotide repeat, thereby reducing or eliminating theexpression or function of aberrant ATXN1 gene products.

The method can further comprise: isolating the mesenchymal stem cellfrom the patient, wherein the mesenchymal stem cell is isolated from thepatient's bone marrow or peripheral blood. The isolating step cancomprise: aspiration of bone marrow and isolation of mesenchymal stemcells using density gradient centrifugation media.

Also provided herein, is an in vivo method for treating a patient withan ATXN1 related disorder. The method can comprise: editing the ATXN1gene in a cell of the patient.

The editing step can comprise: introducing into the cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theATXN1 gene that results in a permanent deletion of the expandedtrinucleotide repeat or replacement of one or more nucleotide bases, orone or more exons and/or introns within or near the ATXN1 gene, therebyrestoring the ATXN1 gene function.

The editing step can comprise: introducing into the cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theATXN1 gene or ATXN1 regulatory elements that results in one or morepermanent insertion, deletion or mutation of at least one nucleotidewithin or near the ATXN1 gene and/or a permanent deletion in theexpanded trinucleotide repeat, thereby reducing or eliminating theexpression or function of aberrant ATXN1 gene products.

The cell can be a cell of the CNS. The cell of the CNS can be a neuron.The cell of the CNS can be a glial cell.

The one or more DNA endonuclease can be delivered to the cell of the CNSvia any administration route selected from the group consisting of:intraparenchymal, intravenous, intra-arterial, intracerebroventricular,intracisternal, intrathecal, intracranial or intraperitoneal routes.

The ATXN1 related condition or disorder can be SCA1.

Also provided herein is a method of altering the contiguous genomicsequence of an ATXN1 gene in a cell. The method can comprise: contactingthe cell with one or more DNA endonuclease to effect one or more SSBs orDSBs. The alteration of the contiguous genomic sequence can occur in oneor more exons of the ATXN1 gene. The alteration of the contiguousgenomic sequence can occur in exon 8 of the ATXN1 gene.

The one or more DNA endonuclease can be selected from any of thosesequences in SEQ ID NOs: 1-620, and variants having at least 90%homology to any of those sequences disclosed in SEQ ID NOs: 1-620.

The one or more DNA endonuclease can be one or more protein orpolypeptide. The one or more protein or polypeptide can be flanked atthe N-terminus, the C-terminus, or both the N-terminus and C-terminus byone or more nuclear localization signals (NLSs). The one or more proteinor polypeptide can be flanked by two NLSs, one NLS located at theN-terminus and the second NLS located at the C-terminus. The one or moreNLSs can be a SV40 NLS. The one or more DNA endonuclease can be one ormore polynucleotide encoding the one or more DNA endonuclease. The oneor more DNA endonuclease can be one or more ribonucleic acid (RNA)encoding the one or more DNA endonuclease. The one or more RNA can beone or more chemically modified RNA. The one or more RNA can bechemically modified in the coding region. The one or more polynucleotideor one or more RNA can be codon optimized.

The methods can further comprise: introducing one or more gRNA or one ormore sgRNA. The one or more gRNA or one or more sgRNA can comprise aspacer sequence that is complementary to a sequence within or near theexpanded trinucleotide repeat in the ATXN1 gene. The one or more gRNA orone or more sgRNA can comprise a spacer sequence that is complementaryto a DNA sequence within or near the ATXN1 gene. The one or more gRNA orone or more sgRNA can comprise a spacer sequence that is complementaryto a sequence flanking the ATXN1 gene or other sequence that encodes aregulatory element of the ATXN1 gene. The one or more gRNA or one ormore sgRNA can comprise a spacer sequence that is complementary to a DNAsequence within or near exon 8 of the ATXN1 gene. The one or more gRNAor one or more sgRNA can be chemically modified. The one or moremodified sgRNAs can comprise three 2′-O-methyl-phosphorothioate residuesat or near each of its 5′ and 3′ ends. The one or more gRNA or one ormore sgRNA can comprise a RNA sequence corresponding to a sequenceselected from the group consisting of SEQ ID NOs: 42685, 42504, 101247,42473, 101253, 101251, 101250, 101085, 101252, 101259, 101139, 101314,42644, 42421, 101103, 42634, 101054, 101319, 101044, 101152, 101320,42637, 101060, 101150, 101062, 42605, 101249, 101316, 101061, 101248,101296, 42686, 101093, 101137, 101236, 101153, 101313, 42451, 101045,42457, 42416, 42474, 42458, 42450, 101117, 42566, 42417, 101151, 42475,101174, 101106, 101069, 101295, 101084, 42449, 101140, 101261, 101196,101136, 101104, 101177, 101308, 42446, 42447, 42635, 101068, 101176, and101064.

The one or more gRNA or one or more sgRNA can be pre-complexed with theone or more DNA endonuclease to form one or more ribonucleoproteins(RNPs). The pre-complexing can involve a covalent attachment of the oneor more gRNA or one or more sgRNA to the one or more DNA endonuclease.The weight ratio of sgRNA to DNA endonuclease in the RNP can be 1:1.

The one or more DNA endonuclease can be formulated in a liposome orlipid nanoparticle. The one or more DNA endonuclease can be formulatedin a liposome or a lipid nanoparticle which also comprises the one ormore gRNA or one or more sgRNA.

The one or more DNA endonuclease can be encoded in an adeno-associatedvirus (AAV) vector particle. The one or more gRNA or one or more sgRNAcan be encoded in an AAV vector particle. The one or more DNAendonuclease can be encoded in an AAV vector particle which also encodesthe one or more gRNA or one or more sgRNA. The AAV vector particle canbe selected from the group consisting of any of those sequencesdisclosed in SEQ ID NOs: 4734-5302 and Table 2.

The methods can further comprise: introducing into the cell a donortemplate comprising at least a portion of the wild-type ATXN1 gene. Theat least a portion of the wild-type ATXN1 gene can comprise one or moresequences selected from the group consisting of an ATXN1 exon, an ATXN1intron, and a sequence comprising an exon:intron junction of ATXN1. Thedonor template can comprise homologous arms to the genomic locus of theATXN1 gene. The donor template can be either a single or double strandedpolynucleotide.

The donor template can be encoded in an AAV vector particle, where theAAV vector particle is selected from the group consisting of any ofthose disclosed in SEQ ID NOs: 4734-5302 and Table 2. The one or morepolynucleotide encoding one or more DNA endonuclease can be formulatedinto a lipid nanoparticle, and the one or more gRNA or one or more sgRNAcan be delivered to the cell ex vivo by electroporation and the donortemplate can be delivered to the cell by an AAV vector. The one or morepolynucleotide encoding one or more DNA endonuclease can be formulatedinto a liposome or lipid nanoparticle which also comprises the one ormore gRNA or one or more sgRNA and the donor template.

The ATXN1 gene can be located on Chromosome 6: 16,299,112-16,761,491(Genome Reference Consortium—GRCh38).

Also provided herein is a single-molecule guide RNA comprising at leasta spacer sequence that is an RNA sequence corresponding to any one ofSEQ ID NOs: 5305-312,293. The single-molecule guide RNA can furthercomprise a spacer extension region. The single-molecule guide RNA canfurther comprise a tracrRNA extension region. The single-molecule guideRNA can be chemically modified.

The single-molecule guide RNA can be pre-complexed with a DNAendonuclease. The DNA endonuclease can be a Cas9 or Cpf1 endonuclease.The Cas9 or Cpf1 endonuclease can be selected from the group consistingof S. pyogenes Cas9, S. aureus Cas9, N. meningitidis Cas9, S.thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, T denticolaCas9, L. bacterium ND2006 Cpf1 and Acidaminococcus sp. BV3L6 Cpf1, andvariants having at least 90% homology to the endonucleases. The Cas9 orCpf1 endonuclease can comprise one or more NLSs. At least one NLS can beat or within 50 amino acids of the amino-terminus of the Cas9 or Cpf1endonuclease and/or at least one NLS can be at or within 50 amino acidsof the carboxy-terminus of the Cas9 or Cpf1 endonuclease.

Also provided herein is a DNA encoding the single-molecule guide RNA.

Also provided herein is a therapeutic comprising at least one or moregRNAs for editing an ATXN1 gene in a cell from a patient with an ATXN1related condition or disorder, the one or more gRNAs comprising a spacersequence selected from the group consisting of nucleic acid sequences inany one of SEQ ID NOs: 5305-312,293 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient with anATXN1 related condition or disorder formed by the method comprising:introducing one or more DNA endonucleases; introducing one or more gRNAor one or more sgRNA for editing a ATXN1 gene; wherein the one or moregRNAs or sgRNAs comprise a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5305-312,293 of theSequence Listing.

The ATXN1 related condition or disorder can be SCA1.

It is understood that the inventions described in this specification arenot limited to the examples summarized in this Summary Various otheraspects are described and exemplified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods disclosed and described in thisspecification can be better understood by reference to the accompanyingfigures, in which:

FIGS. 1A-B depict the type II CRISPR/Cas system;

FIG. 1A is a depiction of the type II CRISPR/Cas system including gRNA;

FIG. 1B is another depiction of the type II CRISPR/Cas system includingsgRNA;

FIGS. 2A-D describe the cutting efficiencies in the range of 0.8-91.8%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2A describes the cutting efficiencies in the range of 70-91.8% ofS. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNA screenin HEK293T cells;

FIG. 2B describes the cutting efficiencies in the range of 39.6-69.8% ofS. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNA screenin HEK293T cells;

FIG. 2C describes the cutting efficiencies in the range of 10.4-39.5% ofS. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNA screenin HEK293T cells;

FIG. 2D describes the cutting efficiencies in the range of 0.8-7% of S.pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNA screen inHEK293T cells; and

FIG. 3 is a graph describing the cutting efficiencies in the range of0.8-91.8% of S. pyogenes gRNAs in HEK293T cells.

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NOs: 1-620 are Cas endonuclease ortholog sequences.

SEQ ID NOs: 621-631 do not include sequences.

SEQ ID NOs: 632-4715 are microRNA sequences.

SEQ ID NOs: 4716-4733 do not include sequences.

SEQ ID NOs: 4734-5302 are AAV serotype sequences.

SEQ ID NO: 5303 is a ATXN1 nucleotide sequence.

SEQ ID NO: 5304 is a gene sequence including 1-5 kilobase pairs upstreamand/or downstream of the ATXN1 gene.

SEQ ID NOs: 5305-7241 are 20 bp spacer sequences for targeting within ornear a ATXN1 gene or other DNA sequence that encodes a regulatoryelement of the ATXN1 gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 7242-11,702 are 20 bp spacer sequences for targeting withinor near a ATXN1 gene or other DNA sequence that encodes a regulatoryelement of the ATXN1 gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 11,703-25,282 are 20 bp spacer sequences for targetingwithin or near a ATXN1 gene or other DNA sequence that encodes aregulatory element of the ATXN1 gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 25,283-37,855 are 20 bp spacer sequences for targetingwithin or near a ATXN1 gene or other DNA sequence that encodes aregulatory element of the ATXN1 gene with a N. meningitidis Cas9endonuclease.

SEQ ID NOs: 37,856-155,355 are 20 bp spacer sequences for targetingwithin or near a ATXN1 gene or other DNA sequence that encodes aregulatory element of the ATXN1 gene with a S. pyogenes Cas9endonuclease.

SEQ ID NOs: 155,356-312,293 are 20 bp spacer sequences for targetingwithin or near a ATXN1 gene or other DNA sequence that encodes aregulatory element of the ATXN1 gene with an Acidaminococcus, aLachnospiraceae, and a Franciscella Novicida Cpf1 endonuclease.

SEQ ID NOs: 312,294-312,323 do not include sequences.

SEQ ID NO: 312,324 is a sample spacer sequence, including the PAM, for aguide RNA (gRNA) for a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 312,325-312,327 show sample sgRNA sequences.

SEQ ID NO: 312,328 shows a known family of homing endonuclease, asclassified by its structure.

DETAILED DESCRIPTION I. Introduction Genome Editing

The present disclosure provides strategies and techniques for thetargeted, specific alteration of the genetic information (genome) ofliving organisms. As used herein, the term “alteration” or “alterationof genetic information” refers to any change in the genome of a cell. Inthe context of treating genetic disorders, alterations may include, butare not limited to, insertion, deletion and correction. As used herein,the term “insertion” refers to an addition of one or more nucleotides ina DNA sequence. Insertions can range from small insertions of a fewnucleotides to insertions of large segments such as a cDNA or a gene.The term “deletion” refers to a loss or removal of one or morenucleotides in a DNA sequence or a loss or removal of the function of agene. In some cases, a deletion can include, for example, a loss of afew nucleotides, an exon, an intron, a gene segment, or the entiresequence of a gene. In some cases, deletion of a gene refers to theelimination or reduction of the function or expression of a gene or itsgene product. This can result from not only a deletion of sequenceswithin or near the gene, but also other events (e.g., insertion,nonsense mutation) that disrupt the expression of the gene. The term“correction” or “corrected” as used herein, refers to a change of one ormore nucleotides of a genome in a cell, whether by insertion, deletionor substitution. Such correction may result in a more favorablegenotypic or phenotypic outcome, whether in structure or function, tothe genomic site which was corrected. One non-limiting example of a“correction” includes the correction of a mutant or defective sequenceto a wild-type sequence which restores structure or function to a geneor its gene product(s). Depending on the nature of the mutation,correction may be achieved via various strategies disclosed herein. Inone non-limiting example, a missense mutation may be corrected byreplacing the region containing the mutation with its wild-typecounterpart. As another example, duplication mutations (e.g., repeatexpansions) in a gene may be corrected by removing the extra sequences.

In some aspects, alterations may also include a gene knock-in, knock-outor knock-down. As used herein, the term “knock-in” refers to an additionof a DNA sequence, or fragment thereof into a genome. Such DNA sequencesto be knocked-in may include an entire gene or genes, may includeregulatory sequences associated with a gene or any portion or fragmentof the foregoing. For example, a cDNA encoding the wild-type protein maybe inserted into the genome of a cell carrying a mutant gene. Knock-instrategies need not replace the defective gene, in whole or in part. Insome cases, a knock-in strategy may further involve substitution of anexisting sequence with the provided sequence, e.g., substitution of amutant allele with a wild-type copy. On the other hand, the term“knock-out” refers to the elimination of a gene or the expression of agene. For example, a gene can be knocked out by either a deletion or anaddition of a nucleotide sequence that leads to a disruption of thereading frame. As another example, a gene may be knocked out byreplacing a part of the gene with an irrelevant sequence. Finally, theterm “knock-down” as used herein refers to reduction in the expressionof a gene or its gene product(s). As a result of a gene knock-down, theprotein activity or function may be attenuated or the protein levels maybe reduced or eliminated.

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner Examples of methods of genome editing describedherein include methods of using site-directed nucleases to cutdeoxyribonucleic acid (DNA) at precise target locations in the genome,thereby creating single-strand or double-strand DNA breaks at particularlocations within the genome. Such breaks can be and regularly arerepaired by natural, endogenous cellular processes, such ashomology-directed repair (HDR) and non-homologous end joining (NHEJ), asreviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015). These twomain DNA repair processes consist of a family of alternative pathways.NHEJ directly joins the DNA ends resulting from a double-strand break,sometimes with the loss or addition of nucleotide sequence, which maydisrupt or enhance gene expression. HDR utilizes a homologous sequence,or donor sequence, as a template for inserting a defined DNA sequence atthe break point. The homologous sequence can be in the endogenousgenome, such as a sister chromatid. Alternatively, the donor can be anexogenous nucleic acid, such as a plasmid, a single-strandoligonucleotide, a double-stranded oligonucleotide, a duplexoligonucleotide or a virus, that has regions of high homology with thenuclease-cleaved locus, but which can also contain additional sequenceor sequence changes including deletions that can be incorporated intothe cleaved target locus. A third repair mechanism can bemicrohomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ,” in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJcan make use of homologous sequences of a few base pairs flanking theDNA break site to drive a more favored DNA end joining repair outcome,and recent reports have further elucidated the molecular mechanism ofthis process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015);Kent et al., Nature Structural and Molecular Biology, Adv. Onlinedoi:10.1038/nsmb.2961(2015); Mateos-Gomez et al., Nature 518, 254-57(2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some instances,it may be possible to predict likely repair outcomes based on analysisof potential microhomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of site-directedpolypeptides, as described and illustrated herein.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondarystructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). Non-limiting examples of Type IICRISPR systems are shown in FIGS. 1A and 1B. The tracrRNA can bemodified by endogenous RNaseIII, and then hybridizes to a crRNA repeatin the pre-crRNA array. Endogenous RNaseIII can be recruited to cleavethe pre-crRNA. Cleaved crRNAs can be subjected to exoribonucleasetrimming to produce the mature crRNA form (e.g., 5′ trimming) ThetracrRNA can remain hybridized to the crRNA, and the tracrRNA and thecrRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNAof the crRNA-tracrRNA-Cas9 complex can guide the complex to a targetnucleic acid to which the crRNA can hybridize. Hybridization of thecrRNA to the target nucleic acid can activate Cas9 for targeted nucleicacid cleavage. The target nucleic acid in a Type II CRISPR system isreferred to as a protospacer adjacent motif (PAM). In nature, the PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to the target nucleic acid. Type II systems (also referred to asNmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B(CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed thatthe CRISPR/Cas9 system is useful for RNA-programmable genome editing,and international patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpf1 is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpf1-associatedCRISPR arrays can be processed into mature crRNAs without therequirement of an additional trans-activating tracrRNA. The Type VCRISPR array can be processed into short mature crRNAs of 42-44nucleotides in length, with each mature crRNA beginning with 19nucleotides of direct repeat followed by 23-25 nucleotides of spacersequence. In contrast, mature crRNAs in Type II systems can start with20-24 nucleotides of spacer sequence followed by about 22 nucleotides ofdirect repeat. Also, Cpf1 can utilize a T-rich protospacer-adjacentmotif such that Cpf1-crRNA complexes efficiently cleave target DNApreceded by a short T-rich PAM, which is in contrast to the G-rich PAMfollowing the target DNA for Type II systems. Thus, Type V systemscleave at a point that is distant from the PAM, while Type II systemscleave at a point that is adjacent to the PAM. In addition, in contrastto Type II systems, Cpf1 cleaves DNA via a staggered DNA double-strandedbreak with a 4 or 5 nucleotide 5′ overhang. Type II systems cleave via ablunt double-stranded break. Similar to Type II systems, Cpf1 contains apredicted RuvC-like endonuclease domain, but lacks a second HNHendonuclease domain, which is in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides aspublished in Fonfara et al., Nucleic Acids Research, 42: 2577-2590(2014). The CRISPR/Cas gene naming system has undergone extensiverewriting since the Cas genes were discovered. Fonfara et al. alsoprovides PAM sequences for the Cas9 polypeptides from various species(see also Table 1 infra).

II. Compositions and Methods of the Disclosure

Editing Strategy

The methods provided herein, regardless of whether a cellular or ex vivoor in vivo method, can involve one or a combination of the following: 1)deleting the entire expanded trinucleotide repeat or a portion thereofin or near the ATXN1 gene, by inducing two double-stranded DNA breaks atboth sides of the expanded region, and replacing with a correctedsequence; 2) deleting the entire expanded trinucleotide repeat or aportion thereof in or near the ATXN1 gene, by inducing onedouble-stranded DNA break proximal to the expanded region, and replacingwith a corrected sequence; 3) replacing the deleted expandedtrinucleotide repeat sequence with a corrected sequence; 4) reducing(knock-down) or eliminating (knock-out) the expression of the mutantATXN1 gene by inducing one or more insertions, deletions or mutationswithin or near the ATXN1 gene or other DNA sequences that encoderegulatory elements of the ATXN1 gene; 5) disrupting or deleting themutant ATXN1 gene and inserting a wild-type ATXN1 gene, a cDNA or aminigene (comprised of one or more exons and optionally one or moreintrons, including natural or synthetic introns) into the ATXN1 genelocus or a safe harbor locus; 6) correcting a point mutation within ornear the ATXN1 gene.

In the first editing strategy, the dual DSB-induced deletion strategycan involve excising the entire abnormal repeat expansion or a portionthereof in the ATXN1 gene, by inducing two or more double strandedbreaks at both sides of the repeat region with one or more CRISPRendonucleases and two or more sgRNAs. In certain aspects, a donor DNAcontaining the corrected sequence can be provided to restore thewild-type sequence. This approach can require development andoptimization of sgRNAs and donor DNA molecules for the ATXN1 gene.

In the second editing strategy, the single DSB-induced deletion strategycan involve deleting the entire abnormal repeat expansion or a portionthereof in the ATXN1 gene by inducing one double stranded break at asite proximal to the repeat region with one or more CRISPR endonucleasesand a gRNA (e.g., crRNA+tracrRNA, or sgRNA). In certain aspects, a donorDNA containing the corrected sequence can be provided to restore thewild-type sequence. This approach can require development andoptimization of gRNAs and donor DNA molecule for the ATXN1 gene.

In the third editing strategy, replacing the deleted expandedtrinucleotide repeat sequence with a corrected sequence can be achievedby delivering into the cell one or more CRISPR endonucleases, pairs ofgRNAs (e.g., crRNA+tracrRNA, or sgRNA) targeting upstream or downstreamof the expanded trinucleotide repeat sequence, and a donor DNA thatcontains the desired sequence and homology arms to the flanking regionsof the target locus. This approach can require development andoptimization of sgRNAs for the ATXN1 gene.

In the fourth editing strategy, the knock-down or knock-out strategy caninvolve disrupting the reading frame in the ATXN1 gene by introducingrandom insertions or deletions (indels) that arise due to the impreciseNHEJ repair pathway. This can be achieved by inducing one singlestranded break or double stranded break in the ATXN1 gene with one ormore CRISPR endonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA),or two or more single stranded breaks or double stranded breaks in theATXN1 gene with two or more CRISPR endonucleases and two or more sgRNAs.This approach can require development and optimization of sgRNAs for theATXN1 gene.

The fifth editing strategy can involve disruption or deletion of themutant ATXN1 gene and insertion of a wild-type ATXN1 gene, a cDNA or aminigene (comprised of one or more exons and optionally one or moreintrons, including natural or synthetic introns) into the locus of theATXN1 gene. In certain aspects, this can be achieved by delivering intothe cell one or more CRISPR endonucleases, a pair of gRNAs (e.g.,crRNA+tracrRNA, or sgRNA) targeting upstream and downstream of or in thefirst and last exon and/or intron of the ATXN1 gene, and a donor DNAthat contains the desired sequence and homology arms to the flankingregions of the target locus. The cytogenetic location of the ATXN1 geneis 6p22.3. Alternatively, the wild-type ATXN1 gene, a cDNA or a minigenecan be inserted into a safe harbor locus. A safe harbor locus refers toa region of the genome where the integrated material can be adequatelyexpressed without perturbing endogenous genome structure or function.The safe harbor loci include but are not limited to AAVS1 (intron 1 ofPPP1R12C), HPRT, H11, hRosa26 and/or F-A region. The safe harbor locican be selected from the group consisting of: exon 1, intron 1 or exon 2of PPP1R12C; exon 1, intron 1 or exon 2 of HPRT; and exon 1, intron 1 orexon 2 of hRosa26. The safe harbor loci may be exons or introns ofubiquitously expressed genes and/or genes with tissue specificexpression (e.g.: nervous system). The whole gene correction strategymay also involve deletion of the endogenous mutated ATXN1 gene inconjunction with insertion of the wild-type ATXN1 gene.

The whole gene correction strategy utilizes a donor DNA template inHomology-Directed Repair (HDR). HDR may be accomplished by making one ormore single-stranded breaks (SSBs) or double-stranded breaks (DSBs) atspecific sites in the genome by using one or more endonucleases. Thedonor DNA can be single or double stranded DNA. The donor template canhave homologous arms to the 6p22.3 region. The donor template can havehomologous arms to a safe harbor locus. For example, the donor templatecan have homologous arms to an AAVS1 safe harbor locus, such as, intron1 of the PPP1R12C gene. As another example, the donor template can havehomologous arms to a hRosa26 safe harbor locus.

In the sixth editing strategy, correcting a point mutation or repeatexpansion can involve replacing one or more nucleotide bases, or one ormore exons and/or introns within or near the ATXN1 gene. Correctingpoint mutation or repeat expansion can involve deleting the sequencecontaining the mutation by inducing a double stranded break at a siteproximal and a site distal to the point mutation with one or more CRISPRendonucleases and a gRNA (e.g., crRNA+tracrRNA, or sgRNA). In certainaspects, a donor DNA containing the corrected sequence can be providedto restore the wild-type sequence. This approach can require developmentand optimization of gRNAs and donor DNA molecule for the ATXN1 gene.

In this way, the editing strategies set forth in the present disclosurecan help to restore the wild-type or similar ATXN1 intronic sequence of,or otherwise reduce or eliminate the expression of the mutant ATXN1 genewith as few as a single treatment (rather than deliver potentialtherapies for the lifetime of the patient).

The advantages for the above strategies are similar, including inprinciple both short and long term beneficial clinical and laboratoryeffects. The whole gene correction approach does provide one advantageover the mutational correction approach—the ability to treat allpatients versus only a subset of patients.

In addition to the above genome editing strategies, another strategyinvolves modulating expression, function, or activity of ATXN1 byediting in the regulatory sequence.

In addition to the editing options listed above, Cas9 or similarproteins can be used to target effector domains to the same target sitesthat can be identified for editing, or additional target sites withinrange of the effector domain A range of chromatin modifying enzymes,methylases or demethylases can be used to alter expression of the targetgene. One possibility is decreasing the expression of the ATXN1 proteinif the mutation leads to undesirable activity. These types of epigeneticregulation have some advantages, particularly as they are limited inpossible off-target effects.

The regulation of transcription and translation implicates a number ofdifferent classes of sites that interact with cellular proteins ornucleotides. Often the DNA binding sites of transcription factors orother proteins can be targeted for mutation or deletion to study therole of the site, though they can also be targeted to change geneexpression. Sites can be added through non-homologous end joining NHEJor direct genome editing by homology directed repair (HDR). Increaseduse of genome sequencing, RNA expression and genome-wide studies oftranscription factor binding have increased our ability to identify howthe sites lead to developmental or temporal gene regulation. Thesecontrol systems can be direct or can involve extensive cooperativeregulation that can require the integration of activities from multipleenhancers. Transcription factors typically bind 6-12 bp-long degenerateDNA sequences. The low level of specificity provided by individual sitessuggests that complex interactions and rules are involved in binding andthe functional outcome. Binding sites with less degeneracy can providesimpler means of regulation. Artificial transcription factors can bedesigned to specify longer sequences that have less similar sequences inthe genome and have lower potential for off-target cleavage. Any ofthese types of binding sites can be mutated, deleted or even created toenable changes in gene regulation or expression (Canver, M. C. et al.,Nature (2015)).

Site-Directed Polypeptides (Endonucleases, Enzymes)

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA. The site-directed polypeptide can be administered to a cellor a patient as either: one or more polypeptides, or one or more mRNAsencoding the polypeptide. Any of the enzymes or orthologs disclosed inSEQ ID NOs: 1-620, or disclosed herein, may be utilized in the methodsherein. Single-molecule guide RNA can be pre-complexed with asite-directed polypeptide. The site-directed polypeptide can be any ofthe DNA endonuclease disclosed herein.

In the context of a CRISPR/Cas9 or CRISPR/Cpf1 system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In the CRISPR/Cas9or CRISPR/Cpf1 systems disclosed herein, the site-directed polypeptidecan be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprise a plurality of nucleicacid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. For example,the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the term“Cas9” refers to both a naturally-occurring and a recombinant Cas9. Cas9enzymes contemplated herein can comprise a HNH or HNH-like nucleasedomain, and/or a RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell's endogenous DNA-repairpathways (e.g., homology-dependent repair (HDR) or NHEJ or alternativenon-homologous end joining (A-NHEJ) or microhomology-mediated endjoining (MMEJ)). NHEJ can repair cleaved target nucleic acid without theneed for a homologous template. This can sometimes result in smalldeletions or insertions (indels) in the target nucleic acid at the siteof cleavage, and can lead to disruption or alteration of geneexpression. HDR can occur when a homologous repair template, or donor,is available. The homologous donor template can comprise sequences thatare homologous to sequences flanking the target nucleic acid cleavagesite. The sister chromatid can be used by the cell as the repairtemplate. However, for the purposes of genome editing, the repairtemplate can be supplied as an exogenous nucleic acid, such as aplasmid, duplex oligonucleotide, single-strand oligonucleotide or viralnucleic acid. With exogenous donor templates, an additional nucleic acidsequence (such as a transgene) or modification (such as a single ormultiple base change or a deletion) can be introduced between theflanking regions of homology so that the additional or altered nucleicacid sequence also becomes incorporated into the target locus. MMEJ canresult in a genetic outcome that is similar to NHEJ in that smalldeletions and insertions can occur at the cleavage site. MMEJ can makeuse of homologous sequences of a few base pairs flanking the cleavagesite to drive a favored end-joining DNA repair outcome. In someinstances, it may be possible to predict likely repair outcomes based onanalysis of potential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a “donorpolynucleotide” (or donor or donor sequence) herein. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. The donorpolynucleotide can be an exogenous polynucleotide sequence, i.e., asequence that does not naturally occur at the target nucleic acidcleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

The site-directed polypeptide can comprise an amino acid sequence havingat least 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% amino acidsequence identity to a wild-type exemplary site-directed polypeptide[e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence ID No. 8 orSapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], andvarious other site-directed polypeptides. The site-directed polypeptidecan comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identityto a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids. The site-directed polypeptide cancomprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids. The site-directed polypeptide cancomprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids in a HNH nuclease domain of thesite-directed polypeptide. The site-directed polypeptide can comprise atmost: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at least: 70,75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at most: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a RuvC nuclease domain of the site-directed polypeptide.

The site-directed polypeptide can comprise a modified form of awild-type exemplary site-directed polypeptide. The modified form of thewild-type exemplary site-directed polypeptide can comprise a mutationthat reduces the nucleic acid-cleaving activity of the site-directedpolypeptide. The modified form of the wild-type exemplary site-directedpolypeptide can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form ofthe site-directed polypeptide can have no substantial nucleicacid-cleaving activity. When a site-directed polypeptide is a modifiedform that has no substantial nucleic acid-cleaving activity, it isreferred to herein as “enzymatically inactive.”

The modified form of the site-directed polypeptide can comprise amutation such that it can induce a single-strand break (SSB) on a targetnucleic acid (e.g., by cutting only one of the sugar-phosphate backbonesof a double-strand target nucleic acid). In some aspects, the mutationcan result in less than 90%, less than 80%, less than 70%, less than60%, less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, or less than 1% of the nucleic acid-cleavingactivity in one or more of the plurality of nucleic acid-cleavingdomains of the wild-type site directed polypeptide (e.g., Cas9 from S.pyogenes, supra). In some aspects, the mutation can result in one ormore of the plurality of nucleic acid-cleaving domains retaining theability to cleave the complementary strand of the target nucleic acid,but reducing its ability to cleave the non-complementary strand of thetarget nucleic acid. The mutation can result in one or more of theplurality of nucleic acid-cleaving domains retaining the ability tocleave the non-complementary strand of the target nucleic acid, butreducing its ability to cleave the complementary strand of the targetnucleic acid. For example, residues in the wild-type exemplary S.pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854 and Asn856, aremutated to inactivate one or more of the plurality of nucleicacid-cleaving domains (e.g., nuclease domains). The residues to bemutated can correspond to residues Asp10, His840, Asn854 and Asn856 inthe wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., asdetermined by sequence and/or structural alignment). Non-limitingexamples of mutations include D10A, H840A, N854A or N856A. One skilledin the art will recognize that mutations other than alaninesubstitutions can be suitable.

In some aspects, a D10A mutation can be combined with one or more ofH840A, N854A, or N856A mutations to produce a site-directed polypeptidesubstantially lacking DNA cleavage activity. A H840A mutation can becombined with one or more of D10A, N854A, or N856A mutations to producea site-directed polypeptide substantially lacking DNA cleavage activity.A N854A mutation can be combined with one or more of H840A, D10A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A N856A mutation can be combined with oneor more of H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domainare referred to as “nickases.”

Nickase variants of RNA-guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ˜20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome—alsoknown as off-target cleavage. Because nickase variants of Cas9 each onlycut one strand, in order to create a double-strand break it is necessaryfor a pair of nickases to bind in close proximity and on oppositestrands of the target nucleic acid, thereby creating a pair of nicks,which is the equivalent of a double-strand break. This requires that twoseparate guide RNAs—one for each nickase—must bind in close proximityand on opposite strands of the target nucleic acid. This requirementessentially doubles the minimum length of homology needed for thedouble-strand break to occur, thereby reducing the likelihood that adouble-strand cleavage event will occur elsewhere in the genome, wherethe two guide RNA sites—if they exist—are unlikely to be sufficientlyclose to each other to enable the double-strand break to form. Asdescribed in the art, nickases can also be used to promote HDR versusNHEJ. HDR can be used to introduce selected changes into target sites inthe genome through the use of specific donor sequences that effectivelymediate the desired changes.

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagine, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

The site-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive site-directedpolypeptide) can target nucleic acid. The site-directed polypeptide(e.g., variant, mutated, enzymatically inactive and/or conditionallyenzymatically inactive endoribonuclease) can target DNA. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., the site-directed polypeptide is a fusion protein).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., a HNH domain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), and a non-native sequence (for example, anuclear localization signal) or a linker linking the site-directedpolypeptide to a non-native sequence.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesmutation of aspartic acid 10, and/or wherein one of the nuclease domainscan comprise a mutation of histidine 840, and wherein the mutationreduces the cleaving activity of the nuclease domain(s) by at least 50%.

The one or more site-directed polypeptides, e.g. DNA endonucleases, cancomprise two nickases that together effect one double-strand break at aspecific locus in the genome, or four nickases that together effect orcause two double-strand breaks at specific loci in the genome.Alternatively, one site-directed polypeptide, e.g. DNA endonuclease, caneffect or cause one double-strand break at a specific locus in thegenome.

Non-limiting examples of Cas9 orthologs from other bacterial strainsinclude but are not limited to, Cas proteins identified in Acaryochlorismarina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacilluscaldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillusacidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifexdegensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328;Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacilluspseudomycoides DSM 12442; Bacillus selenitireducens MLS10;Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725;Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptorhydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridiumdifficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp.PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328;Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp.bulgaricus PB2003/044-T3-4; Lactobacillus salivarius ATCC 11741;Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17;Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01;Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134;Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis;Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp.dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum_SI;Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonassp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomycespristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486;Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736;Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; andThermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5):726-737).

In addition to Cas9 orthologs, other Cas9 variants such as fusionproteins of inactive dCas9 and effector domains with different functionsmay be served as a platform for genetic modulation. Any of the foregoingenzymes may be useful in the present disclosure.

Further examples of endonucleases that may be utilized in the presentdisclosure are provided in SEQ ID NOs: 1-620. These proteins may bemodified before use or may be encoded in a nucleic acid sequence such asa DNA, RNA or mRNA or within a vector construct such as the plasmids orAAV vectors taught herein. Further, they may be codon optimized.

SEQ ID NOs: 1-620 disclose a non-exhaustive listing of endonucleasesequences.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA can comprise at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeatsequence and tracrRNA sequence hybridize to each other to form a duplex.In the Type V guide RNA (gRNA), the crRNA forms a duplex. In bothsystems, the duplex can bind a site-directed polypeptide, such that theguide RNA and site-direct polypeptide form a complex. Thegenome-targeting nucleic acid can provide target specificity to thecomplex by virtue of its association with the site-directed polypeptide.The genome-targeting nucleic acid thus can direct the activity of thesite-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in SEQ ID NOs:5305-312,293 of the Sequence Listing. Each guide RNA can be designed toinclude a spacer sequence complementary to its genomic target sequence.For example, each of the spacer sequences in SEQ ID NOs: 5305-312,293 ofthe Sequence Listing can be put into a single RNA chimera or a crRNA(along with a corresponding tracrRNA). See Jinek et al., Science, 337,816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

The genome-targeting nucleic acid can be a double-molecule guide RNA.The genome-targeting nucleic acid can be a single-molecule guide RNA.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system can comprise, inthe 5′ to 3′ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensioncan comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. The sgRNA can comprise a less than a 20 nucleotidespacer sequence at the 5′ end of the sgRNA sequence. The sgRNA cancomprise a more than 20 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a variable length spacer sequencewith 17-30 nucleotides at the 5′ end of the sgRNA sequence (see Table1).

The sgRNA can comprise no uracil at the 3′end of the sgRNA sequence,such as in SEQ ID NO: 312,326 of Table 1. The sgRNA can comprise one ormore uracil at the 3′end of the sgRNA sequence, such as in SEQ ID NO:312,327 in Table 1. For example, the sgRNA can comprise 1 uracil (U) atthe 3′ end of the sgRNA sequence. The sgRNA can comprise 2 uracil (UU)at the 3′ end of the sgRNA sequence. The sgRNA can comprise 3 uracil(UUU) at the 3′ end of the sgRNA sequence. The sgRNA can comprise 4uracil (UUUU) at the 3′ end of the sgRNA sequence. The sgRNA cancomprise 5 uracil (UUUUU) at the 3′ end of the sgRNA sequence. The sgRNAcan comprise 6 uracil (UUUUUU) at the 3′ end of the sgRNA sequence. ThesgRNA can comprise 7 uracil (UUUUUUU) at the 3′ end of the sgRNAsequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the 3′ end ofthe sgRNA sequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2′-O-methyl phosphorothioate nucleotides.

TABLE 1 SEQ ID NO. sgRNA sequence 312,325nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaa aaguggcaccgagucggugcuuuu 312,326nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaa aaguggcaccgagucggugc 312,327n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgaguc ggugcu₍₁₋₈₎

A single-molecule guide RNA (sgRNA) in a Type V system can comprise, inthe 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacersequence.

By way of illustration, guide RNAs used in the CRISPR/Cas9 orCRISPR/Cpf1 system, or other smaller RNAs can be readily synthesized bychemical means, as illustrated below and described in the art. Whilechemical synthetic procedures are continually expanding, purificationsof such RNAs by procedures such as high performance liquidchromatography (HPLC, which avoids the use of gels such as PAGE) tendsto become more challenging as polynucleotide lengths increasesignificantly beyond a hundred or so nucleotides. One approach used forgenerating RNAs of greater length is to produce two or more moleculesthat are ligated together. Much longer RNAs, such as those encoding aCas9 or Cpf1 endonuclease, are more readily generated enzymatically.Various types of RNA modifications can be introduced during or afterchemical synthesis and/or enzymatic generation of RNAs, e.g.,modifications that enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

Spacer Extension Sequence

In some examples of genome-targeting nucleic acids, a spacer extensionsequence can modify activity, provide stability and/or provide alocation for modifications of a genome-targeting nucleic acid. A spacerextension sequence can modify on- or off-target activity or specificity.In some examples, a spacer extension sequence can be provided. Thespacer extension sequence can have a length of more than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,4000, 5000, 6000, or 7000 or more nucleotides. The spacer extensionsequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,7000 or more nucleotides. The spacer extension sequence can be less than10 nucleotides in length. The spacer extension sequence can be between10-30 nucleotides in length. The spacer extension sequence can bebetween 30-70 nucleotides in length.

The spacer extension sequence can comprise another moiety (e.g., astability control sequence, an endoribonuclease binding sequence, aribozyme). The moiety can decrease or increase the stability of anucleic acid targeting nucleic acid. The moiety can be a transcriptionalterminator segment (i.e., a transcription termination sequence). Themoiety can function in a eukaryotic cell. The moiety can function in aprokaryotic cell. The moiety can function in both eukaryotic andprokaryotic cells. Non-limiting examples of suitable moieties include: a5′ cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence(e.g., to allow for regulated stability and/or regulated accessibilityby proteins and protein complexes), a sequence that forms a dsRNA duplex(i.e., a hairpin), a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid can interactwith a target nucleic acid in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR/Cas system herein, the spacer sequence can be designed tohybridize to a target nucleic acid that is located 5′ of a PAM of theCas9 enzyme used in the system. The spacer may perfectly match thetarget sequence or may have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NRG-3′, where R comprises either A or G, where N is anynucleotide and N is immediately 3′ of the target nucleic acid sequencetargeted by the spacer sequence.

The target nucleic acid sequence can comprise 20 nucleotides. The targetnucleic acid can comprise less than 20 nucleotides. The target nucleicacid can comprise more than 20 nucleotides. The target nucleic acid cancomprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target nucleic acid can comprise at most: 5,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.The target nucleic acid sequence can comprise 20 bases immediately 5′ ofthe first nucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO: 312,324), the target nucleicacid can comprise the sequence that corresponds to the Ns, wherein N isany nucleotide, and the underlined NRG sequence is the S. pyogenes PAM.This target nucleic acid sequence is often referred to as the PAMstrand, and the complementary nucleic acid sequence is often referred tothe non-PAM strand. One of skill in the art would recognize that thespacer sequence hybridizes to the non-PAM strand of the target nucleicacid (FIGS. 1A and 1B).

The spacer sequence that hybridizes to the target nucleic acid can havea length of at least about 6 nucleotides (nt). The spacer sequence canbe at least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 20 nucleotides. In some examples, the spacer sequence cancomprise 19 nucleotides. In some examples, the spacer sequence cancomprise 18 nucleotides. In some examples, the spacer sequence cancomprise 21 nucleotides. In some examples, the spacer sequence cancomprise 22 nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which may bethought of as a bulge or bulges.

The spacer sequence can be designed or chosen using a computer program.The computer program can use variables, such as predicted meltingtemperature, secondary structure formation, predicted annealingtemperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

In some aspects, a minimum CRISPR repeat sequence is a sequence with atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequenceidentity to a reference CRISPR repeat sequence (e.g., crRNA from S.pyogenes).

In some aspects, a minimum CRISPR repeat sequence comprises nucleotidesthat can hybridize to a minimum tracrRNA sequence in a cell. The minimumCRISPR repeat sequence and a minimum tracrRNA sequence can form aduplex, i.e. a base-paired double-stranded structure. Together, theminimum CRISPR repeat sequence and the minimum tracrRNA sequence canbind to the site-directed polypeptide. At least a part of the minimumCRISPR repeat sequence can hybridize to the minimum tracrRNA sequence.At least a part of the minimum CRISPR repeat sequence can comprise atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum tracrRNA sequence. In some aspects, atleast a part of the minimum CRISPR repeat sequence comprises at mostabout 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or 100% complementaryto the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some aspects, the minimum CRISPRrepeat sequence is approximately 9 nucleotides in length. In someaspects, the minimum CRISPR repeat sequence is approximately 12nucleotides in length.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild-type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the minimum CRISPR repeat sequence can be atleast about 65% identical, at least about 70% identical, at least about75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to a reference minimum CRISPR repeat sequence over a stretchof at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

A minimum tracrRNA sequence can comprise nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to a site-directed polypeptide. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

Bulges

In some cases, there can be a “bulge” in the duplex between the minimumCRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region ofnucleotides within the duplex. A bulge can contribute to the binding ofthe duplex to the site-directed polypeptide. The bulge can comprise, onone side of the duplex, an unpaired 5′-XXXY-3′ where X is any purine andY comprises a nucleotide that can form a wobble pair with a nucleotideon the opposite strand, and an unpaired nucleotide region on the otherside of the duplex. The number of unpaired nucleotides on the two sidesof the duplex can be different.

In one example, the bulge can comprise an unpaired purine (e.g.,adenine) on the minimum CRISPR repeat strand of the bulge. In someexamples, the bulge can comprise an unpaired 5′-AAGY-3′ of the minimumtracrRNA sequence strand of the bulge, where Y comprises a nucleotidethat can form a wobble pairing with a nucleotide on the minimum CRISPRrepeat strand.

A bulge on the minimum CRISPR repeat side of the duplex can comprise atleast 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on theminimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3,4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPRrepeat side of the duplex can comprise 1 unpaired nucleotide.

A bulge on the minimum tracrRNA sequence side of the duplex can compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides.A bulge on the minimum tracrRNA sequence side of the duplex can compriseat most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. Abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise 4 unpaired nucleotides.

A bulge can comprise at least one wobble pairing. In some examples, abulge can comprise at most one wobble pairing. A bulge can comprise atleast one purine nucleotide. A bulge can comprise at least 3 purinenucleotides. A bulge sequence can comprise at least 5 purinenucleotides. A bulge sequence can comprise at least one guaninenucleotide. In some examples, a bulge sequence can comprise at least oneadenine nucleotide.

Hairpins

In various examples, one or more hairpins can be located 3′ to theminimum tracrRNA in the 3′ tracrRNA sequence.

The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 or more nucleotides 3′ from the last paired nucleotide in theminimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpincan start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides 3′ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex.

The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. The hairpin can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutivenucleotides.

The hairpin can comprise a CC dinucleotide (i.e., two consecutivecytosine nucleotides).

The hairpin can comprise duplexed nucleotides (e.g., nucleotides in ahairpin, hybridized together). For example, a hairpin can comprise a CCdinucleotide that is hybridized to a GG dinucleotide in a hairpin duplexof the 3′ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some examples, there are two or more hairpins, and in other examplesthere are three or more hairpins.

3′ tracrRNA Sequence

A 3′ tracrRNA sequence can comprise a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

The 3′ tracrRNA sequence can have a length from about 6 nucleotides toabout 100 nucleotides. For example, the 3′ tracrRNA sequence can have alength from about 6 nucleotides (nt) to about 50 nt, from about 6 nt toabout 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The3′ tracrRNA sequence can have a length of approximately 14 nucleotides.

The 3′ tracrRNA sequence can be at least about 60% identical to areference 3′ tracrRNA sequence (e.g., wild type 3′ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3′ tracrRNA sequence can be at least about60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

The 3′ tracrRNA sequence can comprise more than one duplexed region(e.g., hairpin, hybridized region). The 3′ tracrRNA sequence cancomprise two duplexed regions.

The 3′ tracrRNA sequence can comprise a stem loop structure. The stemloop structure in the 3′ tracrRNA can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure inthe 3′ tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nucleotides. The stem loop structure can comprise a functionalmoiety. For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, oran exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the 3′ tracrRNA sequence can comprise a P-domain. In someexamples, the P-domain can comprise a double-stranded region in thehairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence may be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. ThetracrRNA extension sequence can have a length from about 1 nucleotide toabout 400 nucleotides. The tracrRNA extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, or 400 nucleotides. The tracrRNA extension sequence can have alength from about 20 to about 5000 or more nucleotides. The tracrRNAextension sequence can have a length of more than 1000 nucleotides.

The tracrRNA extension sequence can have a length of less than 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. The tracrRNA extension sequence can comprise lessthan 10 nucleotides in length. The tracrRNA extension sequence can be10-30 nucleotides in length. The tracrRNA extension sequence can be30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt. Thefunctional moiety can function in a eukaryotic cell. The functionalmoiety can function in a prokaryotic cell. The functional moiety canfunction in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3′ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). The tracrRNA extension sequence cancomprise a primer binding site or a molecular index (e.g., barcodesequence). The tracrRNA extension sequence can comprise one or moreaffinity tags.

Single-Molecule Guide Linker Sequence

The linker sequence of a single-molecule guide nucleic acid can have alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused, Science, 337(6096):816-821 (2012). An illustrative linker has alength from about 3 nucleotides (nt) to about 90 nt, from about 3 nt toabout 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, fromabout 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3nt to about 10 nt. For example, the linker can have a length from about3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt toabout 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt,from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, fromabout 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90nt to about 100 nt. The linker of a single-molecule guide nucleic acidcan be between 4 and 40 nucleotides. The linker can be at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. The linker can be at most about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used, Science, 337(6096):816-821 (2012), butnumerous other sequences, including longer sequences can likewise beused.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

Nucleic Acid Modifications (Chemical and Structural Modifications)

In some aspects, polynucleotides introduced into cells can comprise oneor more modifications that can be used individually or in combination,for example, to enhance activity, stability or specificity, alterdelivery, reduce innate immune responses in host cells, or for otherenhancements, as further described herein and known in the art.

In certain examples, modified polynucleotides can be used in theCRISPR/Cas9 or CRISPR/Cpf1 system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas9 or Cpf1 endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9 or CRISPR/Cpf1 system toedit any one or more genomic loci.

Using the CRISPR/Cas9 or CRISPR/Cpf1 system for purposes of non-limitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9 or CRISPR/Cpf1genome editing complex comprising guide RNAs, which can besingle-molecule guides or double-molecule, and a Cas9 or Cpf1endonuclease. Modifications of guide RNAs can also or alternatively beused to enhance the initiation, stability or kinetics of interactionsbetween the genome editing complex with the target sequence in thegenome, which can be used, for example, to enhance on-target activity.Modifications of guide RNAs can also or alternatively be used to enhancespecificity, e.g., the relative rates of genome editing at the on-targetsite as compared to effects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in aspects in which a Cas9 orCpf1 endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas9 or Cpf1 endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNases present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9 or CRISPR/Cpf1, forexample, one or more types of modifications can be made to guide RNAs(including those exemplified above), and/or one or more types ofmodifications can be made to RNAs encoding Cas endonuclease (includingthose exemplified above).

By way of illustration, guide RNAs used in the CRISPR/Cas9 orCRISPR/Cpf1 system, or other smaller RNAs can be readily synthesized bychemical means, enabling a number of modifications to be readilyincorporated, as illustrated below and described in the art. Whilechemical synthetic procedures are continually expanding, purificationsof such RNAs by procedures such as high-performance liquidchromatography (HPLC, which avoids the use of gels such as PAGE) tendsto become more challenging as polynucleotide lengths increasesignificantly beyond a hundred or so nucleotides. One approach that canbe used for generating chemically-modified RNAs of greater length is toproduce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 endonuclease, are more readilygenerated enzymatically. While fewer types of modifications areavailable for use in enzymatically produced RNAs, there are stillmodifications that can be used to, e.g., enhance stability, reduce thelikelihood or degree of innate immune response, and/or enhance otherattributes, as described further below and in the art; and new types ofmodifications are regularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2′position of the sugar, in some aspects a 2′-O-alkyl, 2′-O-alkyl-O-alkyl,or 2′-fluoro-modified nucleotide. In some examples, RNA modificationsinclude 2′-fluoro, 2′-amino or 2′-O-methyl modifications on the riboseof pyrimidines, abasic residues, or an inverted base at the 3′ end ofthe RNA. Such modifications are routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone), CH₂—O—N (CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)— CH₂—CH₂ backbones, wherein thenative phosphodiester backbone is represented as O—P—O—CH); amidebackbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch and DavidCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci.,97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂, or O(CH₂)n CH₃, where n is from 1 to about10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some aspects, amodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)) (Martin et al, HeIv. Chim Acta, 1995, 78, 486).Other modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy(2′-OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugarmimetics, such as cyclobutyls in place of the pentofuranosyl group.

In some examples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups. Thebase units can be maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can bereplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases can be retained and bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262. Further teaching of PNA compounds can be foundin Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino) adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil,2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,7-deazaguanine, N6 (6-aminohexyl) adenine, and 2,6-diaminopurine.Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, pp.75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A“universal” base known in the art, e.g., inosine, can also be included.5-Me-C substitutions have been shown to increase nucleic acid duplexstability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu,B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, pp. 276-278) and are aspects of base substitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Further, nucleobases can comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the present disclosure.These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6and 0-6 substituted purines, comprising 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Researchand Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and areaspects of base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588;5,830,653; 6,005,096; and U.S. Patent Application Publication2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can bechemically linked to one or more moieties or conjugates that enhance theactivity, cellular distribution, or cellular uptake of theoligonucleotide. Such moieties comprise, but are not limited to, lipidmoieties such as a cholesterol moiety [Letsinger et al., Proc. Natl.Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al.,Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. AcidsRes., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain[Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)];adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim Biophys.Acta, 1264: 229-237 (1995)]; or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J.Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802;5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963;5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469;5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241;5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785;5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726;5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the present disclosure includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of this presentdisclosure, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this present disclosure, include groups that improveuptake, distribution, metabolism or excretion of the compounds of thepresent disclosure. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23, 1992(published as WO1993007883), and U.S. Pat. No. 6,287,860. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5′ or 3′ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5′ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell's antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5′ cap analogs (such asm7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions(UTRs), or treatment with phosphatase to remove 5′ terminalphosphates—and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead K A et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2′-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2′-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013), and references cited therein.

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide can becodon-optimized according to methods standard in the art for expressionin the cell containing the target DNA of interest. For example, if theintended target nucleic acid is in a human cell, a human codon-optimizedpolynucleotide encoding Cas9 is contemplated for use for producing theCas9 polypeptide.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

Ribonucleoprotein Complexes (RNPs)

The site-directed polypeptide and genome-targeting nucleic acid can eachbe administered separately to a cell or a patient. On the other hand,the site-directed polypeptide can be pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA. Thepre-complexed material can then be administered to a cell or a patient.Such pre-complexed material is known as a ribonucleoprotein particle(RNP). The site-directed polypeptide in the RNP can be, for example, aCas9 endonuclease or a Cpf1 endonuclease. The site-directed polypeptidecan be flanked at the N-terminus, the C-terminus, or both the N-terminusand C-terminus by one or more nuclear localization signals (NLSs). Forexample, a Cas9 endonuclease can be flanked by two NLSs, one NLS locatedat the N-terminus and the second NLS located at the C-terminus. The NLScan be any NLS known in the art, such as a SV40 NLS. The weight ratio ofgenome-targeting nucleic acid to site-directed polypeptide in the RNPcan be 1:1. For example, the weight ratio of sgRNA to Cas9 endonucleasein the RNP can be 1:1.

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, and/or any nucleic acid orproteinaceous molecule necessary to carry out the aspects of the methodsof the disclosure.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, a site-directed polypeptide of the disclosure, and/or anynucleic acid or proteinaceous molecule necessary to carry out theaspects of the methods of the disclosure can comprise a vector (e.g., arecombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector, wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, and those that direct expressionof the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the target cell, the level ofexpression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Other vectors can be used so long as they are compatible with the hostcell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and H1, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., MolecularTherapy—Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector can also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector can also comprise appropriate sequences for amplifyingexpression. The expression vector can also include nucleotide sequencesencoding non-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter). In some cases, the promoter can be a spatially restrictedand/or temporally restricted promoter (e.g., a tissue specific promoter,a cell type specific promoter, etc.).

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure and/or a site-directed polypeptide can be packaged into or onthe surface of delivery vehicles for delivery to cells. Deliveryvehicles contemplated include, but are not limited to, nanospheres,liposomes, quantum dots, nanoparticles, polyethylene glycol particles,hydrogels, and micelles. As described in the art, a variety of targetingmoieties can be used to enhance the preferential interaction of suchvehicles with desired cell types or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

Ex Vivo and In Vivo Based Therapies

Provided herein are methods for treating a patient with Spinocerebellarataxia 1 (SCA1). Spinocerebellar ataxia 1 (SCA1) is caused by abnormalexpansion of a trinucleotide CAG repeat in exon 8 of the ATXN1 gene. Inmost people, the CAG segment is repeated fewer than 39 times. Inpatients with Spinocerebellar ataxia 1 (SCA1), the number of CAG repeatscan range from 40 to more than 80 repeats. This number can be differentfrom one patient to another.

The term “trinucleotide repeat expansion” means a series of three bases(for example, CAG) repeated at least twice. In certain examples, thetrinucleotide repeat expansion may be located in exon 8 of an ATXN1nucleic acid. In certain examples, a pathogenic trinucleotide repeatexpansion includes at least 40 repeats of CAG in an ATXN1 nucleic acidand is associated with disease. In other examples, a pathogenictrinucleotide repeat expansion includes at least 41, 42, 43, 44, 45, 50,55, 60, 65, 70, 75, 80 or more repeats. In certain examples, the repeatsare consecutive. In certain examples, the repeats are interrupted by 1or more nucleobases. In certain examples, a wild-type trinucleotiderepeat expansion includes 39 or fewer repeats of CAG in an ATXN1 nucleicacid. In other examples, a wild-type trinucleotide repeat expansionincludes at most 38, 37, 36, 35, 34, 33, 32, 31, 30, 25, 20, 15, 10, 6,or 4 repeats. In certain examples, the entire trinucleotide repeatexpansion is deleted. In other examples, a portion of the trinucleotiderepeat expansion is deleted.

An aspect of such method is an ex vivo cell-based therapy. For example,a biopsy of the patient's brain is performed. Then, cells of the centralnervous system (e.g., neurons or glial cells) are isolated from thebiopsied material. Then, the chromosomal DNA of the cells of the centralnervous system (e.g., neurons or glial cells) can be edited using thematerials and methods described herein. Finally, the edited cells of thecentral nervous system (e.g., neurons or glial cells) are implanted intothe patient. Any source or type of cell may be used as the progenitorcell.

Another aspect of such method is an ex vivo cell-based therapy. Forexample, a patient specific induced pluripotent stem cell (iPSC) can becreated. Then, the chromosomal DNA of these iPSC cells can be editedusing the materials and methods described herein. Next, thegenome-edited iPSCs can be differentiated into cells, which are thenimplanted into the patient. In certain aspects, the iPSCs can bedifferentiated into cells of the central nervous system (e.g., neurons,glial or retinal cells), cells of the peripheral nervous system or otherperipheral cell types, including cells of the cardiovascular system(e.g.: cardiac myocytes).

Yet another aspect of such method is an ex vivo cell-based therapy. Forexample, a mesenchymal stem cell can be isolated from the patient, whichcan be isolated from the patient's bone marrow or peripheral blood.Next, the chromosomal DNA of these mesenchymal stem cells can be editedusing the materials and methods described herein. Next, thegenome-edited mesenchymal stem cells can be differentiated into cells ofthe central nervous system (e.g., neurons or glial cells). Finally, thedifferentiated cells, e.g., cells of the central nervous system (e.g.,neurons or glial cells) are implanted into the patient.

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. Nuclease-based therapeutics can have some level ofoff-target effects. Performing gene editing ex vivo allows one tocharacterize the edited cell population prior to implantation. Thepresent disclosure includes sequencing the entire genome of the editedcells to ensure that the off-target effects, if any, can be in genomiclocations associated with minimal risk to the patient. Furthermore,populations of specific cells, including clonal populations, can beisolated prior to implantation.

Another advantage of ex vivo cell therapy relates to geneticmodification in iPSCs compared to other primary cell sources. iPSCs areprolific, making it easy to obtain the large number of cells that willbe required for a cell-based therapy. Furthermore, iPSCs are an idealcell type for performing clonal isolations. This allows screening forthe correct genomic modification, without risking a decrease inviability. In contrast, other primary cells, such as glial cells, areviable for only a few passages and difficult to clonally expand. Thus,manipulation of iPSCs for the treatment of Spinocerebellar ataxia 1(SCA1) can be much easier, and can shorten the amount of time needed tomake the desired genetic modification.

Methods can also include an in vivo based therapy. Chromosomal DNA ofthe cells in the patient is edited using the materials and methodsdescribed herein. In some aspects, the target cell in an in vivo basedtherapy is a cell of the central nervous system (e.g., a neuron or aglial cell).

Although certain cells present an attractive target for ex vivotreatment and therapy, increased efficacy in delivery may permit directin vivo delivery to such cells. Ideally the targeting and editing wouldbe directed to the relevant cells. Cleavage in other cells can also beprevented by the use of promoters only active in certain cells and ordevelopmental stages. Additional promoters are inducible, and thereforecan be temporally controlled if the nuclease is delivered as a plasmid.The amount of time that delivered RNA and protein remain in the cell canalso be adjusted using treatments or domains added to change thehalf-life. In vivo treatment would eliminate a number of treatmentsteps, but a lower rate of delivery can require higher rates of editing.In vivo treatment can eliminate problems and losses from ex vivotreatment and engraftment and post-engraftment integration of neuronsand glial cells appropriately into existing brain circuits.

An advantage of in vivo gene therapy can be the ease of therapeuticproduction and administration. The same therapeutic approach and therapywill have the potential to be used to treat more than one patient, forexample a number of patients who share the same or similar genotype orallele. In contrast, ex vivo cell therapy typically requires using apatient's own cells, which are isolated, manipulated and returned to thesame patient.

Also provided herein is a cellular method for editing the ATXN1 gene ina cell by genome editing. For example, a cell can be isolated from apatient or animal. Then, the chromosomal DNA of the cell can be editedusing the materials and methods described herein.

microRNAs (miRNAs)

Another class of gene regulatory regions having these features ismicroRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play keyroles in posttranscriptional gene regulation. miRNA can regulate theexpression of 30% of all mammalian protein-encoding genes. Specific andpotent gene silencing by double stranded RNA (RNAi) was discovered, plusadditional small non-coding RNA (Canver, M. C. et al., Nature (2015)).The largest class of non-coding RNAs important for gene silencing ismiRNAs. In mammals, miRNAs are first transcribed as a long RNAtranscript, which can be separate transcriptional units, part of proteinintrons, or other transcripts. The long transcripts are called primarymiRNA (pri-miRNA) that include imperfectly base-paired hairpinstructures. These pri-miRNA can be cleaved into one or more shorterprecursor miRNAs (pre-miRNAs) by Microprocessor, a protein complex inthe nucleus, involving Drosha.

Pre-miRNAs are short stem loops ˜70 nucleotides in length with a2-nucleotide 3′-overhang that are exported, into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) can be loaded onto the RNA-inducedsilencing complex (RISC). The passenger strand (marked with *), can befunctional, but is usually degraded. The mature miRNA tethers RISC topartly complementary sequence motifs in target mRNAs predominantly foundwithin the 3′ untranslated regions (UTRs) and inducesposttranscriptional gene silencing (Bartel, D. P. Cell 136, 215-233(2009); Saj, A. & Lai, E. C. Curr Opin Genet Dev 21, 504-510 (2011)).

miRNAs can be important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs can also be involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual miRNA transcript can be targeted by many different miRNAs.More than 28645 microRNAs have been annotated in the latest release ofmiRBase (v.21). Some miRNAs can be encoded by multiple loci, some ofwhich can be expressed from tandemly co-transcribed clusters. Thefeatures allow for complex regulatory networks with multiple pathwaysand feedback controls. miRNAs can be integral parts of these feedbackand regulatory circuits and can help regulate gene expression by keepingprotein production within limits (Herranz, H. & Cohen, S. M. Genes Dev24, 1339-1344 (2010); Posadas, D. M. & Carthew, R. W. Curr Opin GenetDev 27, 1-6 (2014)).

miRNA can also be important in a large number of human diseases that areassociated with abnormal miRNA expression. This association underscoresthe importance of the miRNA regulatory pathway. Recent miRNA deletionstudies have linked miRNA with regulation of the immune responses(Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNA also has a strong link to cancer and can play a role in differenttypes of cancer. miRNAs have been found to be downregulated in a numberof tumors. miRNA can be important in the regulation of keycancer-related pathways, such as cell cycle control and the DNA damageresponse, and can therefore be used in diagnosis and can be targetedclinically. MicroRNAs can delicately regulate the balance ofangiogenesis, such that experiments depleting all microRNAs suppressestumor angiogenesis (Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes can also besubject to epigenetic changes occurring with cancer. Many miRNA loci canbe associated with CpG islands increasing their opportunity forregulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B.& Lyko, F. Cell Cycle 6, 1001-1005 (2007)). The majority of studies haveused treatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNA can also activatetranslation (Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out miRNA sites may lead to decreased expression ofthe targeted gene, while introducing these sites may increaseexpression.

Individual miRNA can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the microRNA), which can be important forbinding specificity. Cleavage in this region, followed by mis-repair byNHEJ can effectively abolish miRNA function by blocking binding totarget sites. miRNA could also be inhibited by specific targeting of thespecial loop region adjacent to the palindromic sequence. Catalyticallyinactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. etal., Sci Rep 4, 3943 (2014)). In addition to targeting the miRNA, thebinding sites can also be targeted and mutated to prevent the silencingby miRNA.

According to the present disclosure, any of the microRNA (miRNA) ortheir binding sites may be incorporated into the compositions of thepresent disclosure.

The compositions may have a region such as, but not limited to, a regioncomprising the sequence of any of the microRNAs disclosed in SEQ ID NOs:632-4715, the reverse complement of the microRNAs disclosed in SEQ IDNOs: 632-4715 or the microRNA anti-seed region of any of the microRNAsdisclosed in SEQ ID NOs: 632-4715.

The compositions of the present disclosure may comprise one or moremicroRNA target sequences, microRNA sequences, or microRNA seeds. Suchsequences may correspond to any known microRNA such as those taught inU.S. Publication 2005/0261218 and U.S. Publication 2005/0059005. As anon-limiting example, known microRNAs, their sequences and their bindingsite sequences in the human genome are disclosed in SEQ ID NOs:632-4715.

A microRNA sequence comprises a “seed” sequence, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some aspects, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some aspects, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence.

Identification of microRNA, microRNA target regions, and theirexpression patterns and role in biology have been reported (Bonauer etal., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr OpinHematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413(2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233;Landgraf et al, Cell, 2007 129:1401-1414; Gentner and Naldini, TissueAntigens. 2012 80:393-403).

For example, if the composition is not intended to be delivered to theliver but ends up there, then miR-122, a microRNA abundant in liver, caninhibit the expression of the sequence delivered if one or multipletarget sites of miR-122 are engineered into the polynucleotide encodingthat target sequence. Introduction of one or multiple binding sites fordifferent microRNA can be engineered to further decrease the longevity,stability, and protein translation hence providing an additional layerof tenability.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the compositions of the presentdisclosure, microRNA binding sites can be engineered out of (i.e.removed from) sequences in which they naturally occur in order toincrease protein expression in specific tissues. For example, miR-137binding sites may be removed to improve protein expression in the brain.

Specifically, microRNAs are known to be differentially expressed inimmune cells (also called hematopoietic cells), such as antigenpresenting cells (APCs) (e.g. dendritic cells and macrophages),macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes,natural killer cells, etc Immune cell specific microRNAs are involved inimmunogenicity, autoimmunity, the immune-response to infection,inflammation, as well as unwanted immune response after gene therapy andtissue/organ transplantation Immune cells specific microRNAs alsoregulate many aspects of development, proliferation, differentiation andapoptosis of hematopoietic cells (immune cells). For example, miR-142and miR-146 are exclusively expressed in the immune cells, particularlyabundant in myeloid dendritic cells. Introducing the miR-142 bindingsite into the 3′-UTR of a polypeptide of the present disclosure canselectively suppress the gene expression in the antigen presenting cellsthrough miR-142 mediated mRNA degradation, limiting antigen presentationin professional APCs (e.g. dendritic cells) and thereby preventingantigen-mediated immune response after gene delivery (see, Annoni A etal., blood, 2009, 114, 5152-5161).

In one example, microRNAs binding sites that are known to be expressedin immune cells, in particular, the antigen presenting cells, can beengineered into the polynucleotides to suppress the expression of thepolynucleotide in APCs through microRNA mediated RNA degradation,subduing the antigen-mediated immune response, while the expression ofthe polynucleotide is maintained in non-immune cells where the immunecell specific microRNAs are not expressed.

Many microRNA expression studies have been conducted, and are describedin the art, to profile the differential expression of microRNAs invarious cancer cells/tissues and other diseases. Some microRNAs areabnormally over-expressed in certain cancer cells and others areunder-expressed. For example, microRNAs are differentially expressed incancer cells (WO2008/154098, US2013/0059015, US2013/0042333,WO2011/157294); cancer stem cells (US2012/0053224); pancreatic cancersand diseases (US2009/0131348, US2011/0171646, US2010/0286232, U.S. Pat.No. 8,389,210); asthma and inflammation (U.S. Pat. No. 8,415,096);prostate cancer (US2013/0053264); hepatocellular carcinoma(WO2012/151212, US2012/0329672, WO2008/054828, U.S. Pat. No. 8,252,538);lung cancer cells (WO2011/076143, WO2013/033640, WO2009/070653,US2010/0323357); cutaneous T-cell lymphoma (WO2013/011378); colorectalcancer cells (WO2011/0281756, WO2011/076142); cancer positive lymphnodes (WO2009/100430, US2009/0263803); nasopharyngeal carcinoma(EP2112235); chronic obstructive pulmonary disease (US2012/0264626,US2013/0053263); thyroid cancer (WO2013/066678); ovarian cancer cells(US2012/0309645, WO2011/095623); breast cancer cells (WO2008/154098,WO2007/081740, US2012/0214699), leukemia and lymphoma (WO2008/073915,US2009/0092974, US2012/0316081, US2012/0283310, WO2010/018563).

Non-limiting examples of microRNA sequences and the targeted tissuesand/or cells are disclosed in SEQ ID NOs: 632-4715.

Genome Engineering Strategies

In some aspects, the methods of the present disclosure can involveediting one or both of the mutant alleles. Gene editing to modify orcorrect the ATXN1 gene has the advantage of restoration of correctexpression levels or elimination of aberrant gene products and temporalcontrol.

A step of the ex vivo methods of the present disclosure can compriseediting/correcting the patient specific iPSCs or mesenchymal stem cells.Alternatively, a step of the in vivo methods of the present disclosureinvolves editing/correcting the cells in a Spinocerebellar ataxia 1(SCA1) patient using genome engineering. Similarly, a step in thecellular methods of the present disclosure can compriseediting/correcting the ATXN1 gene in a human cell by genome engineering.

Spinocerebellar ataxia 1 (SCA1) patients exhibit expanded trinucleotiderepeats in the ATXN1 gene. Therefore, different patients may use similarediting strategies. Any CRISPR endonuclease may be used in the methodsof the present disclosure, each CRISPR endonuclease having its ownassociated PAM, which may or may not be disease specific.

In one aspect, the trinucleotide repeat expansion may be excised byinducing two double stranded breaks (DSBs) upstream and downstream ofthe repeat region. Pairs of gRNAs have been used for this type ofdeletions. The CRISPR endonucleases, configured with the two gRNAs,induce two DSBs at the desired locations. After cleavage, the two ends,regardless of whether blunt or with overhangs, can be joined by NHEJ,leading to the deletion of the intervening segment. In certain aspects,the wild-type sequence can be restored by inserting a correct repeatsequence via HDR.

In another aspect, the trinucleotide repeat expansion may be deletedafter inducing a single DSB near or within the repeat region. SingleDSB-induced repeat loss has been reported in several studies includingTALEN-cleaved short CAG/CTG repeats engineered in yeast, ZFN-cleaved CAGrepeats engineered in human cells, and CRISPR/Cas9-cleaved CTG/CAGrepeats engineered in human cells (Richard et al., PLoS ONE (2014),9(4): e95611; Mittelman et al., Proc Natl Acad Sci USA (2009),106(24):9607-12; van Agtmaal et al., Mol Ther (2016),http://dx.doi.org/10.1016/j.ymthe.2016.10.014). A DSB near the repeatregion destabilizes the repeat tracts, triggering a contraction (e.g.partial deletion) or, in some cases, a complete deletion of the repeats.Additionally, work by Mittelman et al. suggested that single DSB-inducedrepeat loss is more effective in longer repeats. In certain aspects, thewild-type sequence can be restored by inserting a correct repeatsequence via HDR.

For example, expression of the mutant ATXN1 gene may be disrupted oreliminated by introducing random insertions or deletions (indels) thatarise due to the imprecise NHEJ repair pathway. The target regions maybe the coding sequence of the ATXN1 gene (i.e., exons). Inserting ordeleting nucleotides into the coding sequence of a gene may cause a“frame shift” where the normal 3-letter codon pattern is disturbed. Inthis way, gene expression and therefore mutant protein production can bereduced or eliminated. This approach may also be used to target anyintron, intron:exon junction, or regulatory DNA element of the ATXN1gene where sequence alteration may interfere with the expression of theATXN1 gene. This approach can require development and optimization ofsgRNAs for the ATXN1 gene.

As a further alternative, the entire mutant gene can be deleted and awild-type gene, a cDNA or a minigene (comprised of one or more exons andintrons or natural or synthetic introns) can be knocked into the genelocus or a heterologous location in the genome such as a safe harborlocus. Pairs of nucleases can be used to delete mutated gene regions,and a donor is provided to restore function. In this case two gRNA andone donor sequence would be supplied. A full length cDNA can be knockedinto any safe harbor locus, but must use a supplied or an endogenouspromoter. If this construct is knocked into the correct location orlocus, it will have physiological control, similar to the normal gene.

Some genome engineering strategies involve correcting the repeatexpansion, or inserting a wild-type ATXN1 gene or cDNA or a minigene(comprised of one or more exons and introns or natural or syntheticintrons) into the locus of the corresponding gene or a safe harbor locusby homology directed repair (HDR), which is also known as homologousrecombination (HR). Homology directed repair can be one strategy fortreating patients that have expanded trinucleotides in the ATXN1 gene.These strategies can restore the ATXN1 gene and reverse, treat, and/ormitigate the diseased state.

Homology directed repair is a cellular mechanism for repairingdouble-stranded breaks (DSBs). The most common form is homologousrecombination. There are additional pathways for HDR, includingsingle-strand annealing and alternative-HDR. Genome engineering toolsallow researchers to manipulate the cellular homologous recombinationpathways to create site-specific modifications to the genome. It hasbeen found that cells can repair a double-stranded break using asynthetic donor molecule provided in trans. Therefore, by introducing adouble-stranded break near a specific mutation and providing a suitabledonor, targeted changes can be made in the genome. Specific cleavageincreases the rate of HDR more than 1,000 fold above the rate of 1 in10⁶ cells receiving a homologous donor alone. The rate of homologydirected repair (HDR) at a particular nucleotide is a function of thedistance to the cut site, so choosing overlapping or nearest targetsites is important. Gene editing offers the advantage over geneaddition, as editing in situ leaves the rest of the genome unperturbed.

Supplied donors for editing by HDR vary markedly but can contain theintended sequence with small or large flanking homology arms to allowannealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides range in size from less than 100 nt to overmany kb, though longer ssDNA can also be generated and used.Double-stranded donors can be used, including PCR amplicons, plasmids,and mini-circles. In general, it has been found that an AAV vector canbe a very effective means of delivery of a donor template, though thepackaging limits for individual donors is <5 kb. Active transcription ofthe donor increased HDR three-fold, indicating the inclusion of promotermay increase conversion. Conversely, CpG methylation of the donordecreased gene expression and HDR.

In addition to wild-type endonucleases, such as Cas9, nickase variantsexist that have one or the other nuclease domain inactivated resultingin cutting of only one DNA strand. HDR can be directed from individualCas nickases or using pairs of nickases that flank the target area.Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection,nano-particle, micro-injection, or viral transduction. A range oftethering options has been proposed to increase the availability of thedonors for HDR. Examples include attaching the donor to the nuclease,attaching to DNA binding proteins that bind nearby, or attaching toproteins that are involved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several non-homologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as alt-NHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. (See e.g.,Maresca, M., Lin, V. G., Guo, N. & Yang, Y., Genome Res 23, 539-546(2013); Cristea et al. Biotechnology and Bioengineering, 110 (3):871-80(2013)); Suzuki et al. Nature, 540, 144-149 (2016)).

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HDR. Acombination approach may be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ may prove effective for ligation inthe intron, while the error-free HDR may be better suited in the codingregion.

The mutation of the ATXN1 gene that causes Spinocerebellar ataxia 1(SCA1) is a trinucleotide repeat expansion of the three letter string ofnucleotides CAG. In healthy individuals, there are few repeats of thistrinucleotide, typically about or fewer than 39. In people with thediseases phenotype, the repeat can occur more than a 100 times. One ormore trinucleotide repeats may be deleted or corrected in order torestore the gene to a wild-type or similar number of trinucleotiderepeats. In some aspects, all of the trinucleotide repeats may bedeleted. Alternatively, the mutant gene may be knocked out to eliminatetoxic gene products. In addition to repeat expansion, point mutationshave also been identified in patients with SCA1. Any one or more of themutations can be repaired in order to restore the ATXN1. In someexamples, one or more nucleotide bases may be replaced to correct thepoint mutation. In other examples, one or more exons and/or intronswithin or near the ATXN1 gene may be replaced. As a further alternative,an ATXN1 gene or cDNA can be inserted to the locus of the correspondinggene to replace the mutant gene or knocked-in to a safe harbor site,such as AAVS1. In some examples, the methods can provide one gRNA or apair of gRNAs that can be used to facilitate incorporation of a newsequence from a polynucleotide donor template to knock-in a part of orthe entire ATXN1 gene or cDNA.

Some genome engineering strategies involves repeat deletion. Targeteddeletion of trinucleotide repeats is an attractive strategy for treatinga large subset of patients with a single therapeutic cocktail. Deletionscan either be single trinucleotide repeat deletions ormulti-trinucleotide repeat deletions. While multi-repeat deletions,including complete deletion of the expanded trinucleotide repeat, canreach a larger number of patients, for larger deletions the efficiencyof deletion greatly decreases with increased size. In some aspects, thedeletions range from 40 to 5,000 base pairs (bp) in size. For example,deletions may range from 40-100; 100-300; 300-500; 500-1,000;1,000-2,000; 2,000-3,000; or 3,000-5,000 base pairs in size.

The methods can provide gRNA pairs that make a deletion by cutting thegene twice, one gRNA cutting at the 5′ end of the trinucleotide repeatsand the other gRNA cutting at the 3′ end of the trinucleotide repeats.The cutting can be accomplished by a pair of DNA endonucleases that eachmakes a DSB in the genome, or by multiple nickases that together make aDSB in the genome.

Alternatively, the methods can provide one gRNA to make onedouble-strand cut around the trinucleotide repeats. The double-strandcut can be made by a single DNA endonuclease or multiple nickases thattogether make a DSB in the genome.

Illustrative modifications within the ATXN1 gene include deletionswithin or near (proximal) to the trinucleotide repeats referred toabove, such as within the region of less than 3 kb, less than 2 kb, lessthan 1 kb, less than 0.5 kb upstream or downstream of the specificmutation. Given the relatively wide variations of trinucleotide repeatsin the ATXN1 gene, it will be appreciated that numerous variations ofthe deletions referenced above (including without limitation larger aswell as smaller deletions), would be expected to result in restorationof the ATXN1 gene expression.

Such variants can include deletions that are larger in the 5′ and/or 3′direction than the specific repeat expansion in question, or smaller ineither direction. Accordingly, by “near” or “proximal” with respect tospecific repeat expansion, it is intended that the SSB or DSB locusassociated with a desired deletion boundary (also referred to herein asan endpoint) can be within a region that is less than about 3 kb fromthe reference locus noted. The SSB or DSB locus can be more proximal andwithin 2 kb, within 1 kb, within 0.5 kb, or within 0.1 kb. In the caseof small deletion, the desired endpoint can be at or “adjacent to” thereference locus, by which it is intended that the endpoint can be within100 bp, within 50 bp, within 25 bp, or less than about 10 bp to 5 bpfrom the reference locus.

In order to ensure that the pre-mRNA is properly processed followingdeletion, the surrounding splicing signals can be deleted. Splicingdonor and acceptors are generally within 100 base pairs of theneighboring intron. In some examples, methods can provide gRNAs that cutapproximately +/−100-3100 bp with respect to each exon/intron junctionof interest.

For any of the genome editing strategies, gene editing can be confirmedby sequencing or PCR analysis.

Target Sequence Selection

Shifts in the location of the 5′ boundary and/or the 3′ boundaryrelative to particular reference loci can be used to facilitate orenhance particular applications of gene editing, which depend in part onthe endonuclease system selected for the editing, as further describedand illustrated herein.

In a first non-limiting example of such target sequence selection, manyendonuclease systems have rules or criteria that can guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another non-limiting example of target sequence selection oroptimization, the frequency of off-target activity for a particularcombination of target sequence and gene editing endonuclease (i.e. thefrequency of DSBs occurring at sites other than the selected targetsequence) can be assessed relative to the frequency of on-targetactivity. In some cases, cells that have been correctly edited at thedesired locus can have a selective advantage relative to other cells.Illustrative, but non-limiting, examples of a selective advantageinclude the acquisition of attributes such as enhanced rates ofreplication, persistence, resistance to certain conditions, enhancedrates of successful engraftment or persistence in vivo followingintroduction into a patient, and other attributes associated with themaintenance or increased numbers or viability of such cells. In othercases, cells that have been correctly edited at the desired locus can bepositively selected for by one or more screening methods used toidentify, sort or otherwise select for cells that have been correctlyedited. Both selective advantage and directed selection methods can takeadvantage of the phenotype associated with the correction. In somecases, cells can be edited two or more times in order to create a secondmodification that creates a new phenotype that is used to select orpurify the intended population of cells. Such a second modificationcould be created by adding a second gRNA enabling expression of aselectable or screenable marker. In some cases, cells can be correctlyedited at the desired locus using a DNA fragment that contains the cDNAand also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection canalso be guided by consideration of off-target frequencies in order toenhance the effectiveness of the application and/or reduce the potentialfor undesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity can be influenced by a number of factorsincluding similarities and dissimilarities between the target site andvarious off-target sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of double-strand breaks (DSBs), which occur on aregular basis during the normal cell replication cycle but can also beenhanced by the occurrence of various events (such as UV light and otherinducers of DNA breakage) or the presence of certain agents (such asvarious chemical inducers). Many such inducers cause DSBs to occurindiscriminately in the genome, and DSBs can be regularly induced andrepaired in normal cells. During repair, the original sequence can bereconstructed with complete fidelity, however, in some cases, smallinsertions or deletions (referred to as “indels”) are introduced at theDSB site.

DSBs can also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that can comprise as few as ten base pairs orless, can also be used to bring about desired deletions. For example, asingle DSB can be introduced at a site that exhibits microhomology witha nearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce deletions orreplacements that result in restoration of wild-type or similar levelsof trinucleotide repeats in the ATXN1 gene, as well as the selection ofspecific target sequences within such regions that are designed tominimize off-target events relative to on-target events.

Human Cells

For ameliorating Spinocerebellar ataxia 1 (SCA1) or any disorderassociated with ATXN1, as described and illustrated herein, theprincipal targets for gene editing are human cells. For example, in theex vivo methods, the human cells can be somatic cells, which after beingmodified using the techniques as described, can give rise todifferentiated cells, e.g., neurons or progenitor cells. For example, inthe in vivo methods, the human cells may be cells from the centralnervous system or cells from other affected organs.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that will beeffective in ameliorating one or more clinical conditions associatedwith the patient's disease.

Stem cells are capable of both proliferation and giving rise to moreprogenitor cells, these in turn having the ability to generate a largenumber of mother cells that can in turn give rise to differentiated ordifferentiable daughter cells. The daughter cells themselves can beinduced to proliferate and produce progeny that subsequentlydifferentiate into one or more mature cell types, while also retainingone or more cells with parental developmental potential. The term “stemcell” refers then, to a cell with the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Inone aspect, the term progenitor or stem cell refers to a generalizedmother cell whose descendants (progeny) specialize, often in differentdirections, by differentiation, e.g., by acquiring completely individualcharacters, as occurs in progressive diversification of embryonic cellsand tissues. Cellular differentiation is a complex process typicallyoccurring through many cell divisions. A differentiated cell may derivefrom a multipotent cell that, itself, is derived from a multipotentcell, and so on. While each of these multipotent cells may be consideredstem cells, the range of cell types that each can give rise to may varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity may benatural or may be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can also be“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

Self-renewal can be another important aspect of the stem cell. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Generally,“progenitor cells” have a cellular phenotype that is more primitive(i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell). Often, progenitorcells also have significant or very high-proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a neural progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a neural precursor), and then to anend-stage differentiated cell, such as a neuron, which plays acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further.

Induced Pluripotent Stem Cells

The genetically engineered human cells described herein can be inducedpluripotent stem cells (iPSCs). An advantage of using iPSCs is that thecells can be derived from the same subject to which the progenitor cellsare to be administered. That is, a somatic cell can be obtained from asubject, reprogrammed to an induced pluripotent stem cell, and thenre-differentiated into a progenitor cell to be administered to thesubject (e.g., autologous cells). Because the progenitors areessentially derived from an autologous source, the risk of engraftmentrejection or allergic response can be reduced compared to the use ofcells from another subject or group of subjects. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one aspect, the stem cells used in the disclosed methods arenot embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells non-differentiatedcells (e.g., undifferentiated cells) or pluripotent cells. Thetransition of a differentiated cell to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming Reprogramming can encompasscomplete reversion of the differentiation state of a differentiated cell(e.g., a somatic cell) to a pluripotent state or a multipotent state.Reprogramming can encompass complete or partial reversion of thedifferentiation state of a differentiated cell (e.g., a somatic cell) toan undifferentiated cell (e.g., an embryonic-like cell). Reprogrammingcan result in expression of particular genes by the cells, theexpression of which further contributes to reprogramming. In certainexamples described herein, reprogramming of a differentiated cell (e.g.,a somatic cell) can cause the differentiated cell to assume anundifferentiated state (e.g., is an undifferentiated cell). Theresulting cells are referred to as “reprogrammed cells,” or “inducedpluripotent stem cells (iPSCs or iPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a myogenic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some examples.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014); andreferences cited therein. The production of iPSCs can be achieved by theintroduction of nucleic acid sequences encoding stem cell-associatedgenes into an adult, somatic cell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it may not be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28.Reprogramming using the methods and compositions described herein canfurther comprise introducing one or more of Oct-3/4, a member of the Soxfamily, a member of the Klf family, and a member of the Myc family to asomatic cell. The methods and compositions described herein can furthercomprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYCand Klf4 for reprogramming. As noted above, the exact method used forreprogramming is not necessarily critical to the methods andcompositions described herein. However, where cells differentiated fromthe reprogrammed cells are to be used in, e.g., human therapy, in oneaspect the reprogramming is not effected by a method that alters thegenome. Thus, in such examples, reprogramming can be achieved, e.g.,without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, an agent or combination of agents that enhance theefficiency or rate of induced pluripotent stem cell production can beused in the production of patient-specific or disease-specific iPSCs.Some non-limiting examples of agents that enhance reprogrammingefficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9ahistone methyltransferase), PD0325901 (a MEK inhibitor), DNAmethyltransferase inhibitors, histone deacetylase (HDAC) inhibitors,valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide,hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g.,6-(3-chlorophenylureido) caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP50. Other reprogramming enhancingagents include, for example, dominant negative forms of the HDACs (e.g.,catalytically inactive forms), siRNA inhibitors of the HDACs, andantibodies that specifically bind to the HDACs. Such inhibitors areavailable, e.g., from BIOMOL International, Fukasawa, Merck Biosciences,Novartis, Gloucester Pharmaceuticals, Titan Pharmaceuticals, MethylGene,and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto,Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one case, for example, acell that expresses Oct4 or Nanog is identified as pluripotent. Methodsfor detecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses.Detection can involve not only RT-PCR, but can also include detection ofprotein markers. Intracellular markers may be best identified viaRT-PCR, or protein detection methods such as immunocytochemistry, whilecell surface markers are readily identified, e.g., byimmunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells can be introduced into nude mice andhistology and/or immunohistochemistry can be performed on a tumorarising from the cells. The growth of a tumor comprising cells from allthree germ layers, for example, further indicates that the cells arepluripotent stem cells.

Cells of the Central Nervous System

In some aspects, the genetically engineered human cells described hereinare cells of the central nervous system. Neurons, which processinformation, and glial cells (or neuroglia), which provide mechanicaland metabolic support to the nervous system and modulate informationprocessed by neurons, are the two main classes of cells of the centralnervous system. Non-limiting examples of neurons include sensory neurons(also referred to as afferent neurons) transfer information from theexternal environment to the central nervous system, motor neurons (alsoreferred to as efferent neurons) transfer information from the centralnervous system to the external environment, and interneurons (alsoreferred to as association neurons) process information in the centralnervous system and transfers the information from one neuron to theother within the central nervous system. Non-limiting examples of glialcells include astrocytes, oligodendrocytes, microglia and Schwann cells.CNS progenitor cells can be neural progenitor cells or glial progenitorcells.

The typical neuron transmits electrical signals from one cell toanother. Neurons contain a cell body, dendrite, axon hillock, axon,nerve endings, neuronal synapses, and neuromuscular junctions. Neuronsmay be named according to shape or the nature of the dendritic tree.

Neuroglia differ from neurons in several general ways in that they: donot form synapses, have essentially only one type of process, retain theability to divide, and are less electrically excitable than neurons.Neuroglia are classified based on size and shape of their nucleus anddistinguished from neurons, at the light microscopic level. Neurogliaare divided into two major categories based on size, the macroglia andthe microglia. The macroglia are of ectodermal origin and consist ofastrocytes, oligodendrocytes and ependymal cells. Microglia cells areprobably of mesodermal origin.

Creating Patient Specific iPSCs

One step of the ex vivo methods of the present disclosure can involvecreating a patient specific iPS cell, patient specific iPS cells, or apatient specific iPS cell line. There are many established methods inthe art for creating patient specific iPS cells, as described inTakahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example,the creating step can comprise: a) isolating a somatic cell, such as askin cell or fibroblast, from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. The set ofpluripotency-associated genes can be one or more of the genes selectedfrom the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18,NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.

Performing a Biopsy or Aspirate of the Patient's Bone Marrow

A biopsy or aspirate is a sample of tissue or fluid taken from the body.There are many different kinds of biopsies or aspirates. Nearly all ofthem involve using a sharp tool to remove a small amount of tissue. Ifthe biopsy will be on the skin or other sensitive area, numbing medicinecan be applied first. A biopsy or aspirate may be performed according toany of the known methods in the art. For example, in a bone marrowaspirate, a large needle is used to enter the pelvis bone to collectbone marrow.

Isolating a Mesenchymal Stem Cell

Mesenchymal stem cells can be isolated according to any method known inthe art, such as from a patient's bone marrow or peripheral blood. Forexample, marrow aspirate can be collected into a syringe with heparin.Cells can be washed and centrifuged on a Percoll. The cells can becultured in Dulbecco's modified Eagle's medium (DMEM) (low glucose)containing 10% fetal bovine serum (FBS) (Pittinger M F, Mackay A M, BeckS C et al., Science 1999; 284:143-147).

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas9 or CRISPR/Cpf1 system). In some ex vivo examples herein,the genetically modified cell can be genetically modified neuralprogenitor cell. In some in vivo examples herein, the geneticallymodified cell can be a genetically modified cell of the central nervoussystem (e.g., a neuron or a glial cell). A genetically modified cellcomprising an exogenous genome-targeting nucleic acid and/or anexogenous nucleic acid encoding a genome-targeting nucleic acid iscontemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of ATXN1 gene orprotein expression or activity, for example Western Blot analysis of theATXN1 protein or real time PCR for quantifying ATXN1 mRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell can be cultured in vitro, e.g., under definedconditions or in the presence of other cells. Optionally, the cell canbe later introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In somecases, the isolated population can be a substantially pure population ofcells, as compared to the heterogeneous population from which the cellswere isolated or enriched. In some cases, the isolated population can bean isolated population of human progenitor cells, e.g., a substantiallypure population of human progenitor cells, as compared to aheterogeneous population of cells comprising human progenitor cells andcells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratingSpinocerebellar ataxia 1 (SCA1).

The term “substantially enriched” with respect to a particular cellpopulation, refers to a population of cells that is at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or morewith respect to the cells making up a total cell population.

The term “substantially pure” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,at least about 85%, at least about 90%, or at least about 95% pure, withrespect to the cells making up a total cell population. That is, theterms “substantially pure” or “essentially purified,” with regard to apopulation of progenitor cells, refers to a population of cells thatcontain fewer than about 20%, about 15%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orless than 1%, of cells that are not progenitor cells as defined by theterms herein.

Differentiation of Genome-Edited iPSCs into Cells of the Central NervousSystem (CNS)

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited iPSCs into cells of thecentral nervous system (CNS) (e.g., neurons or glial cells). Thedifferentiating step may be performed according to any method known inthe art.

Differentiation of Genome-Edited Mesenchymal Stem Cells into Cells ofthe Central Nervous System (CNS)

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited mesenchymal stem cells intocells of the central nervous system (CNS). The differentiating step maybe performed according to any method known in the art.

Implanting Cells into Patients

Another step of the ex vivo methods of the present disclosure involvesimplanting the cells of the central nervous system (e.g., neurons orglial cells) into patients. This implanting step may be accomplishedusing any method of implantation known in the art. For example, thegenetically modified neurons may be administered to the patient viaintraparenchymal, vascular (e.g., intravenous, intra-arterial), orventricular (e.g., intracerebroventricular, intracisternal, intrathecal)routes or other routes such as intracranial or intraperitonealinjection.

III. Formulations and Delivery Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions can contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some cases, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein can be administered asa suspension with a pharmaceutically acceptable carrier. One of skill inthe art will recognize that a pharmaceutically acceptable carrier to beused in a cell composition will not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Guide RNA Formulation

Guide RNAs of the present disclosure can be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNA compositions can beformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some cases, the pHcan be adjusted to a range from about pH 5.0 to about pH 8. In somecases, the compositions can comprise a therapeutically effective amountof at least one compound as described herein, together with one or morepharmaceutically acceptable excipients. Optionally, the compositions cancomprise a combination of the compounds described herein, or can includea second active ingredient useful in the treatment or prevention ofbacterial growth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by viral or non-viral delivery vehiclesknown in the art, such as electroporation or lipid nanoparticles. Infurther alternative aspects, the DNA endonuclease can be delivered asone or more polypeptides, either alone or pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

For polynucleotides of the present disclosure, the formulation may beselected from any of those taught, for example, in InternationalApplication PCT/US2012/069610.

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, may be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle may range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs may be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, may be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid can each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP can be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerousmodifications are known in the art and can be used.

The endonuclease and sgRNA can be generally combined in a 1:1 molarratio. Alternatively, the endonuclease, crRNA and tracrRNA can begenerally combined in a 1:1:1 molar ratio. However, a wide range ofmolar ratios can be used to produce a RNP.

AAV (Adeno Associated Virus)

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived, and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes describedherein. Production of pseudotyped rAAV is disclosed in, for example,international patent application publication number WO 01/83692.

AAV Serotypes

AAV particles packaging polynucleotides encoding compositions of thepresent disclosure, e.g., endonucleases, donor sequences, or RNA guidemolecules, of the present disclosure may comprise or be derived from anynatural or recombinant AAV serotype. According to the presentdisclosure, the AAV particles may utilize or be based on a serotypeselected from any of the following serotypes, and variants thereofincluding but not limited to AAV1, AAV10, AAV106.1/hu.37, AAV11,AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43,AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54,AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60,AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2, AAV2.5T,AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6,AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.50, AAV2-5/rh.51, AAV27.3,AAV29.3/bb.1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3,AAV3.1/hu.6, AAV3.1/hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17,AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4,AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13,AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a,AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20,AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2,AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r11.64, AAV4-8/rh.64,AAV4-9/rh.54, AAV5, AAV52.1/hu.20, AAV52/hu.19, AAV5-22/rh.58,AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27,AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2,AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84,AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAV-b, AAVC1, AAVC2, AAVC5,AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1,AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5,AAV-h, AAVH-1/hu.1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhER1.14,AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5,AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31,AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.11,AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18,AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31,AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40,AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2,AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60,AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8,AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39,AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04,AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11,AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19,AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4,AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10,AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19,AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25,AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36,AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44,AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1,AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52,AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59,AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2,AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73,AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8RR533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprineAAV, Japanese AAV 10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK16,AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVSM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and/or AAV SM 10-8.

In some aspects, the AAV serotype may be, or have, a mutation in theAAV9 sequence as described by N Pulicherla et al. (Molecular Therapy19(6):1070-1078 (2011)), such as but not limited to, AAV9.9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

In some aspects, the AAV serotype may be, or have, a sequence asdescribed in U.S. Pat. No. 6,156,303, such as, but not limited to, AAV3B(SEQ ID NO: 1 and 10 of U.S. Pat. No. 6,156,303), AAV6 (SEQ ID NO: 2, 7and 11 of U.S. Pat. No. 6,156,303), AAV2 (SEQ ID NO: 3 and 8 of U.S.Pat. No. 6,156,303), AAV3A (SEQ ID NO: 4 and 9, of U.S. Pat. No.6,156,303), or derivatives thereof.

In some aspects, the serotype may be AAVDJ or a variant thereof, such asAAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8 maycomprise two or more mutations in order to remove the heparin bindingdomain (HBD). As a non-limiting example, the AAV-DJ sequence describedas SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, may comprise two mutations:(1) R587Q where arginine (R; Arg) at amino acid 587 is changed toglutamine (Q; Gln) and (2) R590T where arginine (R; Arg) at amino acid590 is changed to threonine (T; Thr). As another non-limiting example,may comprise three mutations: (1) K406R where lysine (K; Lys) at aminoacid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R;Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (3) R590Twhere arginine (R; Arg) at amino acid 590 is changed to threonine (T;Thr).

In some aspects, the AAV serotype may be, or have, a sequence asdescribed in International Publication No. WO2015121501, such as, butnot limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501),“UPenn AAV10” (SEQ ID NO: 8 of WO2015121501), “Japanese AAV10” (SEQ IDNO: 9 of WO2015121501), or variants thereof.

According to the present disclosure, AAV capsid serotype selection oruse may be from a variety of species. In one example, the AAV may be anavian AAV (AAAV). The AAAV serotype may be, or have, a sequence asdescribed in U.S. Pat. No. 9,238,800, such as, but not limited to, AAAV(SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Pat. No. 9,238,800),or variants thereof.

In one example, the AAV may be a bovine AAV (BAAV). The BAAV serotypemay be, or have, a sequence as described in U.S. Pat. No. 9,193,769,such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. Pat. No.9,193,769), or variants thereof. The BAAV serotype may be or have asequence as described in U.S. Pat. No. 7,427,396, such as, but notlimited to, BAAV (SEQ ID NO: 5 and 6 of U.S. Pat. No. 7,427,396), orvariants thereof.

In one example, the AAV may be a caprine AAV. The caprine AAV serotypemay be, or have, a sequence as described in U.S. Pat. No. 7,427,396,such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Pat. No.7,427,396), or variants thereof.

In other examples, the AAV may be engineered as a hybrid AAV from two ormore parental serotypes. In one example, the AAV may be AAV2G9 whichcomprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be,or have, a sequence as described in U.S. Patent Publication No.20160017005.

In one example, the AAV may be a serotype generated by the AAV9 capsidlibrary with mutations in amino acids 390-627 (VP1 numbering) asdescribed by Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011). The serotype and corresponding nucleotide and amino acidsubstitutions may be, but is not limited to, AAV9.1 (G1594C; D532H),AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y),AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G,C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A,G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T,A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S),AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T;Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C,Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D),AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N,N98K, V6061), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S),AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T;N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V),9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I),AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A;Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R,A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A;F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H),AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65(C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80(G1441A; G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87(T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G,A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C,C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R,T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

In one example, the AAV may be a serotype comprising at least one AAVcapsid CD8+ T-cell epitope. As a non-limiting example, the serotype maybe AAV1, AAV2 or AAV8.

In one example, the AAV may be a variant, such as PHP.A or PHP.B asdescribed in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.

In one example, the AAV may be a serotype selected from any of thosefound in SEQ ID NOs: 4734-5302 and Table 2.

In one example, the AAV may be encoded by a sequence, fragment orvariant as disclosed in SEQ ID NOs: 4734-5302 and Table 2.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types can be transduced by the indicatedAAV serotypes among others.

TABLE 2 Tissue/Cell Types and Serotypes Tissue/Cell Type Serotype LiverAAV3, AAV5, AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9Central nervous system AAV1, AAV4, AAV5, AAV8, AAV9 RPE AAV5, AAV4,AAV2, AAV8, AAV9, AAVrh8R Photoreceptor cells AAV5, AAV8, AAV9, AAVrh8RLung AAV9, AAV5 Heart AAV8 Pancreas AAV8 Kidney AAV2, AAV8 Hematopoieticstem cells AAV6

In addition to adeno-associated viral vectors, other viral vectors canbe used. Such viral vectors include, but are not limited to, lentivirus,alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, EpsteinBarr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplexvirus.

In some aspects, Cas9 mRNA, sgRNA targeting one or two sites in ATXN1gene, and donor DNA can each be separately formulated into lipidnanoparticles, or are all co-formulated into one lipid nanoparticle.

In some aspects, Cas9 mRNA can be formulated in a lipid nanoparticle,while sgRNA and donor DNA can be delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, asmRNA or as a protein. The guide RNA can be expressed from the same DNA,or can also be delivered as an RNA. The RNA can be chemically modifiedto alter or improve its half-life, or decrease the likelihood or degreeof immune response. The endonuclease protein can be complexed with thegRNA prior to delivery. Viral vectors allow efficient delivery; splitversions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV,as can donors for HDR. A range of non-viral delivery methods also existthat can deliver each of these components, or non-viral and viralmethods can be employed in tandem. For example, nano-particles can beused to deliver the protein and guide RNA, while AAV can be used todeliver a donor DNA.

IV. Dosing and Administration

The terms “administering,” “introducing” and “transplanting” are usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells e.g., progenitor cells, or their differentiatedprogeny can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some aspects described herein, aneffective amount of neural progenitor cells is administered via asystemic route of administration, such as an intraperitoneal orintravenous route.

The terms “individual,” “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some aspects, the subject is amammal. In some aspects, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of Spinocerebellarataxia 1 (SCA1). Accordingly, the prophylactic administration of aprogenitor cell population serves to prevent Spinocerebellar ataxia 1(SCA1).

A progenitor cell population being administered according to the methodsdescribed herein can comprise allogeneic progenitor cells obtained fromone or more donors. Such progenitors may be of any cellular or tissueorigin, e.g., liver, muscle, cardiac, brain, etc. “Allogeneic” refers toa progenitor cell or biological samples comprising progenitor cellsobtained from one or more different donors of the same species, wherethe genes at one or more loci are not identical. For example, a liverprogenitor cell population being administered to a subject can bederived from one more unrelated donor subjects, or from one or morenon-identical siblings. In some cases, syngeneic progenitor cellpopulations can be used, such as those obtained from geneticallyidentical animals, or from identical twins. The progenitor cells can beautologous cells; that is, the progenitor cells are obtained or isolatedfrom a subject and administered to the same subject, i.e., the donor andrecipient are the same.

The term “effective amount” refers to the amount of a population ofprogenitor cells or their progeny needed to prevent or alleviate atleast one or more signs or symptoms of Spinocerebellar ataxia 1 (SCA1),and relates to a sufficient amount of a composition to provide thedesired effect, e.g., to treat a subject having Spinocerebellar ataxia 1(SCA1). The term “therapeutically effective amount” therefore refers toan amount of progenitor cells or a composition comprising progenitorcells that is sufficient to promote a particular effect whenadministered to a typical subject, such as one who has or is at risk forSpinocerebellar ataxia 1 (SCA1). An effective amount would also includean amount sufficient to prevent or delay the development of a symptom ofthe disease, alter the course of a symptom of the disease (for examplebut not limited to, slow the progression of a symptom of the disease),or reverse a symptom of the disease. It is understood that for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using routine experimentation.

For use in the various aspects described herein, an effective amount ofprogenitor cells comprises at least 10² progenitor cells, at least 5×10²progenitor cells, at least 10³ progenitor cells, at least 5×10³progenitor cells, at least 10⁴ progenitor cells, at least 5×10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2×10⁵progenitor cells, at least 3×10⁵ progenitor cells, at least 4×10⁵progenitor cells, at least 5×10⁵ progenitor cells, at least 6×10⁵progenitor cells, at least 7×10⁵ progenitor cells, at least 8×10⁵progenitor cells, at least 9×10⁵ progenitor cells, at least 1×10⁶progenitor cells, at least 2×10⁶ progenitor cells, at least 3×10⁶progenitor cells, at least 4×10⁶ progenitor cells, at least 5×10⁶progenitor cells, at least 6×10⁶ progenitor cells, at least 7×10⁶progenitor cells, at least 8×10⁶ progenitor cells, at least 9×10⁶progenitor cells, or multiples thereof. The progenitor cells can bederived from one or more donors, or can be obtained from an autologoussource. In some examples described herein, the progenitor cells can beexpanded in culture prior to administration to a subject in needthereof.

In some aspects, reduction of the expanded trinucleotide repeats in theATXN1 gene in cells of patients having Spinocerebellar ataxia 1 (SCA1)can be beneficial for ameliorating one or more symptoms of the disease,for increasing long-term survival, and/or for reducing side effectsassociated with other treatments. Upon administration of such cells tohuman patients, the presence of progenitors that have wild-type orsimilar levels of the trinucleotide repeat in the ATXN1gene isbeneficial. In some cases, effective treatment of a subject gives riseto at least about 3%, 5% or 7% transcripts having wild-type or similarlevels of trinucleotide repeat relative to total ATXN1 transcripts inthe treated subject. In some examples, transcripts having wild-type orsimilar levels of trinucleotide repeat will be at least about 10% oftotal ATXN1 transcripts. In some examples, transcripts having wild-typeor similar levels of trinucleotide repeat will be at least about 20% to30% of total ATXN1 transcripts. Similarly, the introduction of evenrelatively limited subpopulations of cells having wild-type levels oftrinucleotide repeat in the ATXN1 gene can be beneficial in variouspatients because in some situations normalized cells will have aselective advantage relative to diseased cells. However, even modestlevels of progenitors with wild-type or similar levels of trinucleotiderepeat in the ATXN1 gene can be beneficial for ameliorating one or moreaspects of Spinocerebellar ataxia 1 (SCA1) in patients. In someexamples, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90% or more of the neural progenitorsin patients to whom such cells are administered have wild-type orsimilar levels of trinucleotide repeat in the ATXN1 gene.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1×10⁴ cells are delivered tothe desired site for a period of time.

In one aspect of the method, the pharmaceutical composition may beadministered via a route such as, but not limited to, enteral (into theintestine), gastroenteral, epidural (into the dura matter), oral (by wayof the mouth), transdermal, peridural, intracerebral (into thecerebrum), intracerebroventricular (into the cerebral ventricles),epicutaneous (application onto the skin), intradermal, (into the skinitself), subcutaneous (under the skin), nasal administration (throughthe nose), intravenous (into a vein), intravenous bolus, intravenousdrip, intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intra-amniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracisternal (within thecisterna magna cerebellomedularis), intracorneal (within the cornea),dental intracornal, intracoronary (within the coronary arteries),intracorporus cavernosum (within the dilatable spaces of the corporuscavernosa of the penis), intradiscal (within a disc), intraductal(within a duct of a gland), intraduodenal (within the duodenum),intradural (within or beneath the dura), intraepidermal (to theepidermis), intraesophageal (to the esophagus), intragastric (within thestomach), intragingival (within the gingivae), intraileal (within thedistal portion of the small intestine), intralesional (within orintroduced directly to a localized lesion), intraluminal (within a lumenof a tube), intralymphatic (within the lymph), intramedullary (withinthe marrow cavity of a bone), intrameningeal (within the meninges),intramyocardial (within the myocardium), intraocular (within the eye),intraovarian (within the ovary), intrapericardial (within thepericardium), intrapleural (within the pleura), intraprostatic (withinthe prostate gland), intrapulmonary (within the lungs or its bronchi),intrasinal (within the nasal or periorbital sinuses), intraspinal(within the vertebral column), intrasynovial (within the synovial cavityof a joint), intratendinous (within a tendon), intratesticular (withinthe testicle), intrathecal (within the cerebrospinal fluid at any levelof the cerebrospinal axis), intrathoracic (within the thorax),intratubular (within the tubules of an organ), intratumor (within atumor), intratympanic (within the aurus media), intravascular (within avessel or vessels), intraventricular (within a ventricle), iontophoresis(by means of electric current where ions of soluble salts migrate intothe tissues of the body), irrigation (to bathe or flush open wounds orbody cavities), laryngeal (directly upon the larynx), nasogastric(through the nose and into the stomach), occlusive dressing technique(topical route administration, which is then covered by a dressing thatoccludes the area), ophthalmic (to the external eye), oropharyngeal(directly to the mouth and pharynx), parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(within the respiratory tract by inhaling orally or nasally for local orsystemic effect), retrobulbar (behind the pons or behind the eyeball),intramyocardial (entering the myocardium), soft tissue, subarachnoid,subconjunctival, submucosal, topical, transplacental (through or acrossthe placenta), transtracheal (through the wall of the trachea),transtympanic (across or through the tympanic cavity), ureteral (to theureter), urethral (to the urethra), vaginal, caudal block, diagnostic,nerve block, biliary perfusion, cardiac perfusion, photopheresis andspinal.

Modes of administration include injection, infusion, instillation,and/or ingestion. “Injection” includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In some examples, the route is intravenous. Forthe delivery of cells, administration by injection or infusion can bemade.

The cells can be administered systemically. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof Spinocerebellar ataxia 1 (SCA1) can be determined by the skilledclinician. However, a treatment is considered “effective treatment,” ifany one or all of the signs or symptoms of, as but one example, levelsof trinucleotide repeat in the ATXN1 gene are altered in a beneficialmanner (e.g., decreased by at least 10%), or other clinically acceptedsymptoms or markers of disease are improved or ameliorated. Efficacy canalso be measured by failure of an individual to worsen as assessed byhospitalization or need for medical interventions (e.g., progression ofthe disease is halted or at least slowed). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human, or a mammal)and includes: (1) inhibiting the disease, e.g., arresting, or slowingthe progression of symptoms; or (2) relieving the disease, e.g., causingregression of symptoms; and (3) preventing or reducing the likelihood ofthe development of symptoms.

The treatment according to the present disclosure can ameliorate one ormore symptoms associated with Spinocerebellar ataxia 1 (SCA1) byreducing the number of trinucleotide repeat in the ATXN1 gene in theindividual.

V. Features and Properties of the Spinocerebellar Ataxia Type 1 Protein(ATXN1) Gene

ATXN1 has been associated with diseases and disorders such as, but notlimited to, Alzheimer's Disease, Amyotrophic Lateral Sclerosis, Ataxia,Mental disorders, Bipolar Disorder, Brain Neoplasms, Malignant neoplasmof breast, Cerebellar Ataxia, Deglutition Disorders, Presenile dementia,Dystonia, Fanconi Anemia, Cardiac Arrest, Heart failure, Congestiveheart failure, Huntington Disease, Intestinal Polyps, Ischemia,leukemia, Myelocytic Acute Leukemia, Myeloid Chronic Leukemia, AcutePromyelocytic Leukemia, Machado-Joseph Disease, Multiple Myeloma,Myotonic Dystrophy, Neoplasm Metastasis, Neoplasms Vascular Tissue,nervous system disorder, Neuromuscular Diseases, Nystagmus, Obesity,Olivopontocerebellar Atrophies, Parkinson Disease, Polycythemia,Prostatic Neoplasms, Restless Legs Syndrome, Retinal Degeneration,Schizoaffective Disorder, Schizophrenia, Shy-Drager Syndrome,Spinocerebellar Degeneration, Thymoma, Tobacco Use Disorder, VascularDiseases, Aggressive Fibromatosis, Ataxia Spinocerebellar, MyocardialIschemia, Muscle Weakness, Adenocarcinoma of lung (disorder), Ataxic,Cerebral atrophy, Cerebellar degeneration, Spastic syndrome, Atrophic,Malignant neoplasm of prostate, Multiple System Atrophy, Pallidoluysiandegeneration, Dystonia Disorders, Neuropathy, Dementia,Neurodegenerative Disorders, Carcinogenesis, Leukemogenesis, Prostatecarcinoma, Hashimoto Disease, Progressive Neoplastic Disease, Decreasedtendon reflex, Cerebellar atrophy, 5q-syndrome,Dentatorubral-Pallidoluysian Atrophy, Spinocerebellar Ataxia Type 1,Spinocerebellar Ataxia Type 2, Spinocerebellar Ataxia Type 6 (disorder),Atrophy Muscular Spinobulbar, Progressive cerebellar degeneration,Genomic Instability, Attention deficit hyperactivity disorder,Myelodysplastic Syndrome with Isolated del(5q), Creatinine finding,Episodic ataxia type 2 (disorder), Chromosome 5 trisomy 5q, AmyotrophicLateral Sclerosis Sporadic, Neuronal intranuclear inclusion disease,Fanconi Anemia Complementation Group A (disorder), Progressive cGVHD,and Intellectual Disability. Editing the ATXN1 gene using any of themethods described herein may be used to treat, prevent and/or mitigatethe symptoms of the diseases and disorders described herein.

The activity of ATXN1 is largely confined to the central nervous systemand ATXN1 is associated with Spinocerebellar ataxia type 1 (SCA1).Spinocerebellar ataxia type 1 (SCA1) is an inherited disease (autosomaldominant) caused by expansion of a trinucleotide CAG repeat in the ATXN1gene. This CAG repeat region is located in exon 8 of the ATXN1 gene andencodes a polyglutamine tract. In most people, the CAG trinucleotide isrepeated 4 to 39 times. In individuals with SCA1, the number of the CAGrepeat ranges from 40 to more than 80 in most cells. The number ofrepeats in the gene correlates with the age of onset and severity of thedisease. People with 40 to 50 repeats tend to develop signs and symptomsof SCA1 in mid-adulthood, while people with more than 70 repeats usuallyhave signs and symptoms by their teens. The abnormal repeat expansionleads to the production of an altered version of the ATXN1 protein,which aggregates with other proteins in the nucleus, causing toxicity toneuronal cells.

The location of the repeat region can vary depending on alternatesplicing of the RNA transcript. For example, Variant 1 (RefSeqNM_000332) mRNA has 9 exons and the repeat region is in exon 8, whileVariant 2 mRNA (RefSeq NM_001128164) has 8 exons and the repeat regionis located in exon 7.

Spinocerebellar ataxia type 1 (SCA1) affects 1-9 in 100,000 peopleworldwide with significant geographical and ethnic variations. Commonsymptoms of Spinocerebellar ataxia type 1 (SCA1) include optic atrophy,ophthalmoplegia, bulbar and extrapyramidal signs, peripheral neuropathy,and dementia. Previously there was no known cure for spinocerebellarataxia, which is considered to be a progressive and irreversible diseasecausing varying severities of disability. Treatments are generallydirected toward alleviating symptoms, not curing the disease itself.Another option for treating Spinocerebellar ataxia type 1 (SCA1) isphysical therapy to maintain postural balance and gait.

In one example, the target tissue for the compositions and methodsdescribed herein is central nervous system tissue.

In one example, the gene is Spinocerebellar Ataxia Type 1 Protein(ATXN1) which may also be referred to as Ataxin 1, SpinocerebellarAtaxia Type 1 Protein, ATX1, SCA1, Spinocerebellar Ataxia 1(Olivopontocerebellar Ataxia 1, Autosomal Dominant, Ataxin 1),Alternative Ataxin1, and D6S504E. ATXN1 has a cytogenetic location of6p22.3 and the genomic coordinate are on Chromosome 6 on the forwardstrand at position 16,299,112-16,761,491. The nucleotide sequence ofATXN1 is shown as SEQ ID NO: 5303. NUP153 is the gene upstream of ATXN1on the reverse strand and RP3-467D16.3 is the gene downstream of ATXN1on the reverse strand. RP1-151F17.1 is the gene located on the reversestrand opposite of ATXN1. AL034375.1 and AL009031.1 are completelycontained within ATXN1 on the same strand. ATXN1 has a NCBI gene ID of6310, Uniprot ID of P54253 and Ensembl Gene ID of ENSG00000124788. ATXN1has 23,514 SNPs, 24 introns and 27 exons. The exon identifier fromEnsembl and the start/stop sites of the introns and exons are shown inTable 3.

TABLE 3 Exons and Introns for ATXN1 Exon Intron Intron based No. Exon IDStart/Stop No. on Exon ID Start/Stop EX1  ENSE00001168411 16,761,456-INT1  Intron ENSE00001168411- 16,761,297- 16,761,298 ENSE0000127975616,754,092 EX2  ENSE00001279756 16,754,091- INT2  IntronENSE00001279756- 16,754,042- 16,754,043 ENSE00003524794 16,753,348 EX3 ENSE00003524794 16,753,347- INT3  Intron ENSE00003524794- 16,753,232-16,753,233 ENSE00001279744 16,657,902 EX4  ENSE00001279744 16,657,901-INT4  Intron ENSE00001279744- 16,657,775- 16,657,776 ENSE0000127973616,585,908 EX5  ENSE00001279736 16,585,907- INT5  IntronENSE00001279736- 16,585,779- 16,585,780 ENSE00001279728 16,522,689 EX6 ENSE00001279728 16,522,688- INT6  Intron ENSE00001279728- 16,522,626-16,522,627 ENSE00001771379 16,486,110 EX7  ENSE00001771379 16,486,109-INT7  Intron ENSE00001771379- 16,485,971- 16,485,972 ENSE0000084809716,328,471 EX8  ENSE00000848097 16,328,470- INT8  IntronENSE00000848097- 16,326,393- 16,326,394 ENSE00001279802 16,306,860 EX9 ENSE00001279802 16,306,859- INT9  Intron ENSE00003587866- 16,761,297-16,299,112 ENSE00003524794 16,753,348 EX10 ENSE00003587866 16,761,490-INT10 Intron ENSE00003576454- 16,761,297- 16,761,298 ENSE0000352479416,753,348 EX11 ENSE00003576454 16,761,476- INT11 IntronENSE00003524794- 16,753,232- 16,761,298 ENSE00001949008 16,739,919 EX12ENSE00001949008 16,739,918- INT12 Intron ENSE00001906705- 16,761,297-16,736,851 ENSE00001849671 16,739,919 EX13 ENSE00001906705 16,761,491-INT13 Intron ENSE00001840014- 16,761,297- 16,761,298 ENSE0000352479416,753,348 EX14 ENSE00001849671 16,739,918- INT14 IntronENSE00001279744- 16,657,775- 16,738,465 ENSE00001279736 16,585,908 EX15ENSE00001840014 16,761,460- INT15 Intron ENSE00001279728- 16,522,626-16,761,298 ENSE00001833985 16,486,110 EX16 ENSE00001833985 16,486,109-INT16 Intron ENSE00001815697- 16,749,975- 16,485,135 ENSE0000127974416,657,902 EX17 ENSE00001815697 16,750,092- INT17 IntronENSE00001771379- 16,485,971- 16,749,976 ENSE00001848092 16,433,097 EX18ENSE00001848092 16,433,096- INT18 Intron ENSE00001848092- 16,432,945-16,432,946 ENSE00001839179 16,432,734 EX19 ENSE00001839179 16,432,733-INT19 Intron ENSE00001901715- 16,753,232- 16,432,564 ENSE0000127974416,657,902 EX20 ENSE00001901715 16,753,308- INT20 IntronENSE00001279744- 16,657,775- 16,753,233 ENSE00001941808 16,615,626 EX21ENSE00001941808 16,615,625- INT21 Intron ENSE00001168411- 16,761,297-16,614,975 ENSE00001873002 16,667,553 EX22 ENSE00001168411 16,761,456-INT22 Intron ENSE00001925182- 16,761,297- 16,761,298 ENSE0000127975616,754,092 EX23 ENSE00001873002 16,667,552- INT23 IntronENSE00003524794- 16,753,232- 16,666,968 ENSE00001812188 16,739,919 EX24ENSE00001925182 16,761,463- INT24 Intron ENSE00001823855- 16,761,297-16,761,298 ENSE00001868241 16,753,348 EX25 ENSE00001812188 16,739,918-16,739,602 EX26 ENSE00001823855 16,761,479- 16,761,298 EX27ENSE00001868241 16,753,347- 16,752,962

Table 4 provides information on all of the transcripts for the ATXN1gene based on the Ensembl database. Provided in Table 4 are thetranscript ID from Ensembl and corresponding NCBI RefSeq ID for thetranscript, the translation ID from Ensembl and the corresponding NCBIRefSeq ID for the protein, the biotype of the transcript sequence asclassified by Ensembl and the exons and introns in the transcript basedon the information in Table 3.

TABLE 4 Transcript Information for ATXN1 Transcript Protein TranscriptNCBI Translation NCBI Sequence Exon ID from Intron ID from ID RefSeq IDID RefSeq ID Biotype Table 3 Table 3 ENST0000 NM_000332 ENSP00000NP_000323 Protein EX1, EX2, EX3, INT1, INT2, INT3, 0244769.8 244769coding EX4, EX5, EX6, INT4, INT5, INT6, EX7, EX8, EX9 INT7, INT8ENST0000 — — — Processed EX4, EX5, EX6, INT4, INTS, INT6, 0483591.6transcript EX7, EX17, INT16, INT17, EX18, EX19 INT18 ENST0000 — — —Processed EX2, EX3, EX24, INT2, INT22, 0492857.1 transcript EX25 INT23ENST0000 — — — Processed EX3, EX11, INT10, INT11 0467008.5 transcriptEX12 ENST0000 — — — Processed EX13, EX14 INT12 0498374.1 transcriptENST0000 — — — Processed EX26, EX27 INT24 0479680.1 transcript ENST0000— — — Processed EX4, EX20, INT19, INT20 0495178.1 transcript EX21ENST0000 — — — Processed EX3, EX4, EX5, INT3, INT4, INT5, 0473388.6transcript EX6, EX15, INT13, INT15 EX16 ENST0000 — — — Processed EX1,EX23 INT21 0483954.1 transcript ENST0000 NM_001128164 ENSP00000NP_001121636 Protein EX3, EX4, EX5, INT3, INT4, INT5, 0436367.5 416360coding EX6, EX7, EX8, INT6, INT7, INT8, EX9, EX10 INT9

ATXN1 has 23,514 SNPs and the NCBI rs number and/or UniProt VAR numberfor this ATXN1 gene rs2073517, rs2073516, rs2073515, rs2073514,rs2073513, rs2072737, rs2072736, rs2038018, rs2031185, rs2018299,rs2016498, rs2014559, rs2014558, rs2005994, rs2005993, rs2005992,rs1997716, rs1983291, rs1967660, rs1935900, rs1886349, rs1877768,rs1830695, rs1757692, rs1757691, rs1757690, rs1757689, rs1757688,rs1745989, rs1737848, rs1737847, rs1737846, rs1737844, rs1737843,rs1737309, rs1737308, rs1736537, rs1736536, rs1709173, rs1709172,rs1709171, rs1709169, rs1709166, rs1621888, rs1621091, rs1570488,rs1570487, rs1555063, rs1543359, rs1535602, rs1535601, rs1535600,rs1535018, rs1535017, rs1514330, rs1514329, rs1514328, rs1476465,rs1476464, rs1407830, rs1399220, rs1399219, rs1359067, rs1324543,rs1323317, rs1262918, rs1262917, rs1262916, rs1240584, rs1240583,rs1240582, rs1150639, rs1150638, rs1150637, rs1150636, rs1150635,rs649266, rs648411, rs639621, rs633586, rs631661, rs622201, rs611900,rs611512, rs607138, rs598908, rs581812, rs576889, rs570917, rs563125,rs562760, rs559386, rs555430, rs547491, rs532735, rs531089, rs522591,rs500803, rs493352, rs492412, rs478157, rs452163, rs445677, rs439533,rs438374, rs438258, rs437975, rs436356, rs434905, rs432986, rs432761,rs432321, rs431539, rs421172, rs418011, rs413374, rs404468, rs404160,rs404143, rs403834, rs395065, rs393740, rs373748, rs371594, rs370961,rs369988, rs369787, rs366303, rs236982, rs236981, rs236980, rs236979,rs236978, rs236977, rs236976, rs236975, rs236974, rs236973, rs236972,rs236971, rs236970, rs236969, rs236968, rs236967, rs236966, rs236965,rs236964, rs236963, rs236962, rs236961, rs236960, rs236959, rs236958,rs236957, rs236956, rs236955, rs236954, rs236953, rs236952, rs236951,rs236950, rs236949, rs236948, rs236947, rs236946, rs236945, rs236944,rs236943, rs236942, rs236941, rs236940, rs236939, rs236938, rs236937,rs236936, rs236935, rs236934, rs236933, rs236932, rs236931, rs236930,rs236929, rs236928, rs236927, rs236926, rs236925, rs236924, rs236923,rs236922, rs236921, rs236920, rs235162, rs235161, rs235160, rs235159,rs235158, rs235157, rs235156, rs235155, rs235154, rs235153, rs235152,rs235151, rs235150, rs235149, rs235148, rs235147, rs235146, rs192103,rs190208, rs189347, rs189274, rs182932, rs182919, rs180302, rs180017,rs180015, rs180014, rs180013, rs180012, rs180011, rs180010, rs180009,rs180008, rs180007, rs180006, rs180005, rs180004, rs180003, rs180002,rs180001, rs180000, rs179998, rs179997, rs179996, rs179995, rs179994,rs179993, rs179992, rs179991, rs179990, rs179989, rs179988, rs179987,rs179986, rs179985, rs179984, rs179983, rs3828865, rs179982, rs179981,rs3828868, rs179980, rs179979, rs179978, rs179977, rs179976, rs179975,rs179974, rs179973, rs179972, rs179971, rs179970, rs179969, rs179968,rs179967, rs179966, rs179965, rs179964, rs179963, rs179962, rs179961,rs2073518, rs2073519, rs2075974, rs2078846, rs2092298, rs2092299,rs2097655, rs2106351, rs2137873, rs2137874, rs2137875, rs2143067,rs2143452, rs2143453, rs2143454, rs2146753, rs2175894, rs2179574,rs2181542, rs2188369, rs2206255, rs2209026, rs2237162, rs2237163,rs2237164, rs2237165, rs2237166, rs2237167, rs2237168, rs2237169,rs2237170, rs2237171, rs2237172, rs2237173, rs2237174, rs2237175,rs2237176, rs2237180, rs2237181, rs2237182, rs2237183, rs2237184,rs2237185, rs2237186, rs2237187, rs2237188, rs2237189, rs2237190,rs2237191, rs2237192, rs2237193, rs2237194, rs2237195, rs2237196,rs2237197, rs2237198, rs2237199, rs2237200, rs2237201, rs2237202,rs2237203, rs2237204, rs2237205, rs2237206, rs2237207, rs2237208,rs2237209, rs2237210, rs2237211, rs2237212, rs2237213, rs2237214,rs2237215, rs2237216, rs2237217, rs2237218, rs1150634, rs1150633,rs1150632, rs1150631, rs1150630, rs1150629, rs1150628, rs1150627,rs1150626, rs1150625, rs1144700, rs1144699, rs1144698, rs1144697,rs1144696, rs1144695, rs1144694, rs1144693, rs1143886, rs1115820,rs1049197, rs1049196, rs1049189, rs1034592, rs1018421, rs1018388,rs1018387, rs1016223, rs1016222, rs1015568, rs1014984, rs1012207,rs1009056, rs1008252, rs1008251, rs1003824, rs1003384, rs1001211,rs1001210, rs981459, rs962998, rs932412, rs932411, rs932295, rs910046,rs909788, rs909787, rs909786, rs909522, rs881405, rs876607, rs797097,rs760884, rs760883, rs760882, rs760881, rs756782, rs756781, rs756780,rs756779, rs753325, rs742568, rs742567, rs742328, rs732409, rs729771,rs729770, rs729769, rs729768, rs727854, rs723754, rs723054, rs723053,rs721010, rs721009, rs720006, rs719421, rs719420, rs719316, rs719315,rs707851, rs707850, rs707849, rs707848, rs707847, rs707846, rs707845,rs707844, rs697739, rs697738, rs679716, rs666215, rs660586, rs651702,rs2237219, rs2237220, rs2237221, rs2237223, rs2237224, rs2237225,rs2281152, rs2282829, rs2282830, rs2282831, rs2282832, rs2282833,rs2282834, rs2282835, rs2282837, rs2282838, rs2282839, rs2282840,rs2282841, rs2282842, rs2282843, rs2282844, rs2282847, rs2299060,rs2299061, rs2299062, rs2299063, rs2299064, rs2299065, rs2299066,rs2299067, rs2299068, rs2299069, rs2299070, rs2299071, rs2299072,rs2299073, rs2299074, rs2299075, rs2299076, rs2299077, rs2299078,rs2299079, rs2299080, rs2299081, rs2299082, rs2299083, rs2299084,rs2299085, rs2299086, rs2299087, rs2299088, rs2299089, rs2301594,rs2327959, rs2327979, rs2327980, rs2327981, rs2327982, rs2327995,rs2327996, rs2327997, rs2327998, rs2671402, rs2671403, rs2671404,rs2671405, rs2671407, rs2744412, rs2744413, rs2744414, rs2744415,rs2876420, rs2876421, rs3072200, rs3072201, rs3072202, rs3072203,rs3072217, rs3072308, rs3072311, rs3072316, rs3072319, rs3116712,rs3134582, rs3216218, rs3216219, rs3216220, rs3749932, rs3753091,rs3753092, rs3753093, rs3753094, rs3762612, rs3765273, rs3765274,rs3793095, rs3793096, rs3793098, rs3793099, rs3793100, rs3793101,rs3793102, rs3793103, rs3793104, rs3793106, rs3793107, rs3793108,rs3793111, rs3793112, rs3793113, rs3793114, rs3793116, rs3793118,rs3793119, rs3806120, rs3806125, rs3806127, rs3806128, rs3806130,rs3806134, rs3806135, rs3806136, rs3806137, rs3806138, rs3806139,rs3806140, rs3806141, rs3806142, rs3806144, rs3806145, rs3806147,rs3806152, rs3806154, rs3812189, rs3812190, rs3812192, rs3812193,rs3812194, rs3812195, rs3812196, rs3812198, rs3812199, rs3812200,rs3812202, rs3812203, rs3812204, rs3812205, rs3812206, rs3812207,rs3812208, rs3812209, rs3812213, rs3812216, rs3812217, rs3812218,rs3812220, rs3812221, rs3812222, rs3812225, rs3812227, rs3817753,rs3817836, rs3817837, rs3819400, rs3819401, rs3819402, rs3819403,rs3819405, rs3828863, rs3828864, rs3828866, rs3828867, rs3828870,rs3828872, rs3828874, rs3831514, rs3831515, rs3831516, rs3834854,rs3838683, rs3840417, rs3840418, rs3840419, rs3841158, rs3842614,rs3842615, rs3842616, rs3891679, rs4052878, rs179960, rs179959,rs179958, rs179957, rs179956, rs179955, rs179954, rs179953, rs179952,rs179951, rs179950, rs179949, rs179948, rs179947, rs179946, rs179945,rs179944, rs179943, rs179942, rs179941, rs179940, rs179939, rs179938,rs173139, rs171056, rs171055, rs171054, rs171053, rs142517, rs140803,rs111168, rs90671, rs72630, rs168854, rs9477081, rs3401, rs2927,rs4052889, rs4052890, rs4052891, rs4052892, rs4052893, rs4276509,rs4317413, rs4431439, rs4509125, rs4565296, rs4590274, rs4626414,rs4712283, rs4712284, rs4712286, rs4712287, rs4712291, rs4712292,rs4712293, rs4716060, rs4716063, rs4716064, rs4716066, rs4716067,rs4716068, rs4716069, rs4716070, rs4716071, rs5874556, rs5874558,rs5874559, rs5874560, rs5874561, rs5874564, rs5874565, rs5874566,rs5874569, rs5874575, rs5874576, rs6149447, rs6459468, rs6459469,rs6459470, rs6459471, rs6459472, rs6459473, rs6459474, rs6459475,rs6459476, rs6459477, rs6459478, rs6459479, rs6459480, rs6459481,rs6459482, rs6459483, rs6459484, rs6899321, rs6899526, rs6899699,rs6899788, rs6900454, rs6900694, rs6900697, rs6900710, rs6901150,rs6902007, rs6902174, rs6902665, rs6903850, rs6903987, rs6905648,rs6906713, rs6906830, rs6907320, rs6907417, rs6909206, rs6909231,rs6909736, rs6910569, rs6910595, rs6911453, rs6912044, rs6912665,rs6913045, rs6913332, rs6914035, rs6914509, rs6915093, rs6915310,rs6915446, rs6915458, rs6915772, rs6915824, rs6916034, rs6916598,rs6916602, rs6920318, rs6920639, rs6921486, rs6921608, rs6922185,rs6923528, rs6923746, rs6924861, rs6924939, rs6925592, rs6925658,rs6927762, rs6928225, rs6928232, rs6928290, rs6928962, rs6929468,rs6929906, rs6930762, rs6931635, rs6932102, rs6932569, rs6933265,rs6933822, rs6934233, rs6935573, rs6935615, rs6937740, rs6938433,rs6938989, rs6939612, rs6941134, rs6941480, rs6942078, rs7356896,rs7356897, rs7450287, rs7453227, rs7454781, rs7454917, rs7738187,rs7738608, rs7739138, rs7739556, rs7739829, rs7741594, rs7742245,rs7742265, rs7743513, rs7743620, rs7744103, rs7745262, rs7745276,rs7745583, rs7746500, rs7746649, rs7746693, rs7746864, rs7747080,rs7748037, rs7748065, rs7748113, rs7748239, rs7748637, rs7749322,rs7749341, rs7749506, rs7749925, rs7750263, rs7750866, rs7751517,rs7753715, rs7755599, rs7756396, rs7757085, rs7758453, rs7758530,rs7759862, rs7760476, rs7761845, rs7762230, rs7762645, rs7763179,rs7763246, rs7763366, rs7763682, rs7763707, rs7764112, rs7764158,rs7764974, rs7765024, rs7765262, rs7766262, rs7767755, rs7767866,rs7768276, rs7768298, rs7769397, rs7769399, rs7769955, rs7770062,rs7770397, rs7771631, rs7771856, rs7772066, rs7772172, rs7773920,rs7774168, rs7774332, rs7774490, rs7774508, rs7775127, rs7775798,rs7776292, rs9297009, rs9297010, rs9297011, rs9297012, rs9297013,rs9297014, rs9297015, rs9297016, rs9350005, rs9350006, rs9350007,rs9350008, rs9350009, rs9350013, rs9350014, rs9350015, rs9358089,rs9358090, rs9358093, rs9358095, rs9358098, rs9358099, rs9358102,rs9358103, rs9358104, rs9367911, rs9367912, rs9367913, rs9367916,rs9367917, rs9367918, rs9367919, rs9367921, rs9367922, rs9367923,rs9367924, rs9367925, rs9367926, rs9370887, rs9370888, rs9370889,rs9370890, rs9370891, rs9370893, rs9370894, rs9370897, rs9370898,rs9370899, rs9370916, rs9370917, rs9370918, rs9370919, rs9370920,rs9383152, rs9383153, rs9383154, rs9383156, rs9383157, rs9383158,rs9383164, rs9383165, rs9383172, rs9383175, rs9383178, rs9383179,rs9383180, rs9383184, rs9383187, rs9383196, rs9383197, rs9383200,rs9383201, rs9383202, rs9383203, rs9396662, rs9396663, rs9396664,rs9396665, rs9396666, rs9396667, rs9396668, rs9396669, rs9396672,rs9396674, rs9396676, rs9396677, rs9396678, rs9396679, rs9396680,rs9396681, rs9396682, rs9396683, rs9396684, rs9396686, rs9396687,rs9396688, rs9396691, rs9396702, rs9396703, rs9396704, rs9396705,rs9396706, rs9396707, rs9396708, rs9396709, rs9464878, rs9464879,rs9464880, rs9464881, rs9464882, rs9464883, rs9464884, rs9464885,rs9464886, rs9464887, rs9464888, rs9464889, rs9464890, rs9464891,rs9464892, rs9464893, rs9464895, rs9464896, rs9464897, rs9464898,rs9464899, rs9464900, rs9464901, rs4052879, rs4052880, rs4052887,rs4052888, rs9464902, rs9464903, rs9464904, rs9464905, rs9464906,rs9464907, rs9464908, rs9464909, rs9464910, rs9464912, rs9464913,rs9464914, rs9464915, rs9464917, rs9464918, rs9464919, rs9464922,rs9464923, rs9464924, rs9464925, rs9464926, rs9464927, rs9464928,rs9464929, rs9464930, rs9464931, rs9464932, rs9464933, rs9464934,rs9464935, rs9464936, rs9464937, rs9464941, rs9477087, rs9477088,rs9477091, rs9477093, rs9477094, rs9477095, rs9477096, rs9477097,rs9477098, rs9477100, rs9477101, rs9477102, rs9477103, rs9477104,rs9477105, rs9477106, rs9477107, rs9477108, rs9477109, rs9477110,rs9477111, rs9477112, rs9477113, rs9477114, rs9477115, rs9477116,rs9477117, rs9477118, rs4716072, rs4716073, rs4716074, rs4716075,rs4716076, rs4716077, rs4716079, rs4716080, rs4716081, rs4716082,rs4716083, rs4716084, rs4716085, rs4716088, rs4716089, rs4716090,rs4716091, rs4716092, rs5874552, rs5874553, rs5874555, rs9477140,rs9477141, rs9477142, rs9477143, rs9477144, rs9477145, rs9477146,rs9477147, rs9477148, rs9477149, rs9477150, rs9477151, rs9477152,rs9477153, rs9477154, rs9477155, rs9477156, rs9477157, rs9477158,rs9477159, rs9477163, rs9477164, rs9477166, rs9477167, rs9477168,rs9477169, rs9477170, rs9477171, rs9477173, rs9477174, rs9477175,rs9477176, rs9477177, rs9477178, rs9477179, rs9477180, rs9477181,rs9477182, rs9477183, rs9477184, rs9477187, rs9477188, rs9477189,rs9477190, rs9477191, rs9477192, rs9477193, rs9477194, rs9477195,rs9477196, rs9477197, rs9477199, rs9477201, rs9477202, rs9477203,rs9477204, rs9477205, rs9477206, rs9477207, rs9477209, rs9477212,rs9632494, rs9689725, rs9766121, rs9766707, rs9766975, rs9885624,rs10046108, rs10080748, rs10081137, rs10155678, rs10223634, rs10434861,rs10456203, rs10456782, rs10456783, rs10456784, rs10456785, rs10456786,rs10456788, rs10456789, rs10456790, rs10456791, rs10484360, rs10484361,rs10484362, rs10484363, rs10484364, rs10484365, rs10484366, rs10484367,rs10527935, rs10528588, rs10530931, rs10551173, rs10553568, rs10588280,rs10617781, rs10618388, rs10629214, rs10645263, rs10650162, rs10689647,rs10693149, rs10701437, rs10701438, rs10716233, rs10719124, rs10807591,rs10807595, rs10807596, rs10949352, rs10949353, rs10949354, rs10949355,rs10949356, rs10949357, rs10949359, rs10949361, rs10949363, rs10949365,rs10949370, rs10949371, rs10949372, rs10949374, rs10949375, rs10949377,rs10949378, rs10949379, rs11272204, rs11274092, rs11285936, rs11289596,rs11302453, rs11310261, rs11311429, rs11313002, rs11324781, rs11326648,rs11339069, rs11344034, rs11356086, rs11371272, rs11374047, rs11390101,rs11403124, rs11405981, rs11415112, rs11453731, rs11751880, rs11752067,rs11753506, rs11753508, rs11754283, rs11754317, rs11754363, rs11754792,rs11754856, rs11754887, rs11754994, rs11755219, rs11755448, rs11756606,rs11756866, rs11757530, rs11757883, rs11757955, rs11758500, rs11759105,rs11759487, rs11759688, rs11759913, rs11760177, rs11961079, rs11962254,rs11965783, rs11965878, rs11967040, rs11968098, rs11969151, rs119696124,rs11969836, rs11970452, rs11970726, rs12055437, rs12110539, rs12110544,rs12110809, rs12111246, rs12171545, rs12174561, rs12174717, rs12176384,rs12177565, rs12178213, rs12181605, rs12182481, rs12182692, rs12191215,rs12192158, rs12192388, rs12192838, rs12193455, rs12194346, rs12194553,rs12194640, rs12194917, rs12195401, rs12195609, rs12196135, rs12196585,rs12196785, rs12196800, rs12196949, rs12197334, rs12197536, rs12198202,rs12198703, rs12198838, rs12200127, rs12200396, rs12200687, rs12201800,rs12203162, rs12203546, rs12203830, rs12204397, rs12204510, rs12204969,rs12205143, rs12207277, rs12208093, rs12208490, rs12210729, rs12211010,rs12211562, rs12211723, rs12211759, rs12212310, rs12213096, rs12214500,rs12214893, rs12215395, rs12216364, rs12332862, rs12332928, rs12332929,rs12332930, rs12333014, rs12333133, rs12333185, rs12524151, rs12524184,rs12524208, rs12524527, rs12524663, rs12524796, rs12524970, rs12524984,rs12525391, rs12526264, rs12526924, rs12526926, rs12527059, rs12527135,rs12527194, rs12527937, rs12528322, rs12529114, rs12529351, rs12529415,rs12529751, rs12660074, rs12660125, rs12660297, rs12660684, rs12660756,rs12660858, rs12661684, rs12662293, rs12662315, rs12663557, rs12664028,rs12664074, rs12664438, rs12664454, rs12665181, rs12665284, rs12665423,rs12665660, rs13190752, rs13191231, rs13192226, rs13192653, rs13192772,rs13193106, rs13193481, rs13193551, rs13195696, rs13195909, rs13196679,rs13196683, rs13196807, rs13198559, rs13199994, rs9477082, rs9477083,rs9477085, rs13211000, rs13213072, rs13214592, rs13214602, rs13214750,rs13214776, rs13214787, rs13215730, rs13215732, rs13216386, rs13216812,rs13216884, rs13217365, rs13218013, rs13218023, rs13218161, rs13219554,rs13219690, rs13219866, rs13220378, rs13437136, rs13437167, rs13437181,rs9477119, rs9477120, rs9477121, rs9477122, rs9477123, rs9477124,rs9477127, rs9477128, rs9477129, rs9477130, rs9477131, rs9477133,rs9477134, rs9477135, rs9477136, rs9477137, rs9477138, rs9477139,rs16878841, rs16878842, rs16878866, rs16878879, rs16878884, rs16878891,rs16878898, rs16878902, rs16878924, rs16878937, rs16878941, rs16878946,rs16878970, rs16879003, rs16879006, rs16879008, rs16879023, rs16879064,rs16879071, rs17591931, rs17592806, rs17593451, rs17593746, rs17595094,rs17603856, rs17606174, rs17606216, rs17607371, rs17615335, rs17651247,rs17651969, rs17664365, rs17667167, rs17668407, rs17669102, rs17669205,rs17669356, rs17670414, rs17670444, rs17670763, rs17671883, rs17672487,rs28363693, rs28416217, rs28423057, rs28431830, rs57395401, rs57446118,rs28435888, rs28436936, rs28463035, rs28476552, rs28480387, rs28489166,rs28555263, rs28623772, rs28629304, rs28633299, rs28642106, rs28655232,rs28674533, rs28678013, rs28695570, rs28715150, rs28721935, rs28723595,rs28807989, rs33911700, rs33939841, rs33989686, rs34005468, rs34012238,rs34013429, rs34047670, rs34048626, rs34066790, rs34068811, rs34070370,rs34088655, rs34092584, rs34115253, rs34124550, rs34144915, rs34156851,rs34157279, rs34178988, rs34183749, rs34185158, rs34197922, rs34227296,rs34230333, rs34251579, rs34265178, rs34270442, rs34273547, rs34275283,rs34280987, rs34282758, rs34293751, rs34296275, rs34301676, rs34302009,rs34322250, rs34324423, rs34339699, rs34362098, rs34370975, rs34385461,rs34390620, rs34394515, rs34428316, rs34444826, rs34452089, rs34453220,rs34454648, rs34468480, rs34475975, rs34476774, rs34479386, rs34481749,rs34510488, rs34515868, rs34521638, rs34533789, rs34536223, rs34537223,rs34546632, rs34554293, rs34555740, rs34556179, rs34563300, rs34587095,rs34592722, rs34603917, rs34607092, rs34620895, rs34627413, rs34639141,rs34656657, rs34659387, rs34662176, rs34663670, rs34675197, rs34684208,rs34704575, rs34705242, rs34707694, rs34720078, rs34730383, rs34741864,rs34756673, rs34769591, rs34785399, rs34794258, rs34794431, rs34808546,rs34810455, rs34812556, rs34817813, rs34833835, rs34860003, rs34924784,rs34939814, rs34964163, rs34976621, rs34987043, rs35004713, rs35012753,rs35048991, rs35049086, rs35052561, rs35062254, rs35065615, rs35078278,rs35083248, rs35094506, rs35106041, rs35139131, rs35186034, rs35187181,rs35197098, rs35210572, rs35211918, rs35212899, rs35216501, rs35242778,rs35256389, rs35259243, rs35261121, rs35291830, rs35300321, rs35302881,rs35303561, rs35303612, rs35308265, rs35309467, rs35316106, rs35341350,rs35352184, rs35373534, rs35384376, rs35392206, rs35397431, rs35405868,rs35407192, rs35410078, rs35427312, rs35428239, rs35432282, rs35447898,rs35449480, rs35451965, rs35460025, rs35460656, rs35463858, rs35469421,rs35472967, rs35476061, rs35505523, rs35505747, rs35511971, rs35525606,rs35531117, rs35531937, rs35542349, rs35561504, rs35568512, rs35572190,rs35576346, rs35592530, rs35593294, rs35596834, rs35611774, rs35617797,rs35620088, rs35644007, rs35644456, rs35658454, rs35659505, rs35660875,rs35661479, rs35670885, rs35680579, rs35683923, rs35690453, rs35693428,rs35698667, rs35703487, rs35708539, rs35713988, rs35732274, rs35745288,rs35749735, rs35783435, rs35794731, rs35800888, rs35840220, rs35840871,rs35848249, rs35859660, rs35868875, rs35877501, rs35884389, rs35893990,rs35922163, rs35923930, rs35940923, rs35946151, rs35948236, rs35951981,rs35952639, rs35953770, rs35958457, rs35966888, rs35976793, rs35997021,rs35998417, rs36008949, rs36013922, rs36016138, rs36017353, rs36019158,rs36021268, rs36025980, rs36048068, rs36053300, rs13201064, rs13202625,rs13203090, rs13203108, rs13203213, rs13203214, rs13203215, rs13203218,rs13203471, rs13203584, rs13203793, rs13204889, rs13205339, rs13206084,rs13206449, rs13206823, rs13207781, rs13207955, rs13207978, rs13208159,rs13208160, rs13208251, rs13209167, rs13210207, rs13210989, rs55700377,rs62389044, rs62389045, rs55722776, rs55724467, rs55739923, rs55803813,rs55815465, rs55835653, rs55840780, rs55860911, rs55892705, rs55938980,rs55957812, rs55985124, rs55992872, rs56011259, rs56015325, rs56035456,rs56036913, rs56041438, rs56070209, rs56071504, rs56072347, rs56073887,rs13437182, rs13437332, rs16877817, rs16877822, rs16877885, rs16877977,rs16877981, rs16877998, rs16878011, rs16878043, rs16878121, rs16878129,rs16878179, rs16878307, rs16878314, rs16878335, rs16878358, rs16878425,rs16878431, rs16878435, rs16878453, rs16878479, rs16878486, rs16878668,rs16878682, rs16878684, rs56085766, rs56096746, rs56105499, rs56128107,rs56142053, rs56144015, rs56171990, rs56175042, rs56178525, rs56180657,rs56183437, rs56192497, rs56200963, rs56208125, rs56244538, rs56262935,rs56275358, rs56275788, rs16878688, rs16878696, rs16878698, rs16878709,rs16878721, rs16878723, rs16878738, rs16878791, rs56277549, rs56284322,rs56323642, rs56332149, rs56336401, rs56337026, rs56350922, rs56355703,rs56374431, rs56384477, rs56393937, rs56404385, rs56412324, rs56412348,rs56706428, rs56723656, rs56776996, rs56785042, rs56792852, rs56851169,rs56892354, rs56927851, rs56937975, rs56942732, rs56970591, rs56975695,rs56979917, rs56981561, rs56997662, rs57015402, rs57015912, rs57033937,rs57034032, rs57084115, rs57103427, rs57113835, rs57119969, rs57191682,rs57213813, rs57218395, rs57242101, rs57275122, rs57349714, rs57381204,rs57383878, rs57464015, rs57468874, rs57484346, rs57523148, rs57543605,rs57546158, rs57547112, rs57551111, rs57572612, rs57588833, rs57597775,rs57611705, rs57612700, rs57630664, rs57633877, rs57656320, rs57669062,rs57681677, rs57688789, rs57705650, rs57752763, rs57755389, rs57763404,rs57791752, rs57823516, rs57836342, rs57858152, rs57917864, rs57939953,rs57980871, rs57987667, rs58000476, rs58013909, rs58028904, rs58041497,rs58048762, rs58059754, rs58082907, rs58095172, rs58127383, rs58129263,rs58216803, rs58223053, rs58241493, rs58286188, rs58374104, rs58388037,rs58423054, rs58463209, rs58542930, rs59766671, rs59793332, rs59796086,rs59844886, rs59876926, rs59919885, rs59940147, rs59989031, rs60000645,rs60028007, rs60077871, rs60080010, rs60084677, rs60089226, rs60138312,rs60209783, rs60212716, rs60231131, rs60238072, rs60261388, rs60281559,rs60303929, rs60335523, rs60352927, rs60377551, rs60385649, rs60396177,rs60400915, rs60416047, rs60417047, rs60418833, rs60425642, rs60436225,rs60494656, rs60506317, rs60508345, rs60534627, rs60535095, rs60544205,rs60573597, rs60582286, rs60586256, rs60592002, rs60608169, rs60676338,rs60679834, rs60689654, rs60694744, rs60725859, rs60753753, rs60773492,rs60773814, rs60792431, rs60809031, rs60818889, rs60830508, rs60843390,rs60852545, rs60865965, rs60896264, rs60903586, rs60903636, rs60930957,rs60968679, rs60996338, rs61006601, rs61061856, rs61068397, rs61075502,rs61082256, rs61131185, rs61177706, rs61193572, rs61228681, rs61267105,rs61295131, rs61312254, rs61319398, rs61364171, rs61369372, rs61398920,rs61406228, rs61418119, rs61430899, rs61455187, rs61455630, rs61463158,rs61481495, rs61601003, rs61610206, rs61623707, rs61649104, rs61682278,rs61683267, rs61689184, rs61689758, rs61696661, rs61710662, rs61745116,rs61747470, rs61888822, rs62387702, rs62387706, rs62387707, rs62387708,rs62387710, rs62387741, rs62387742, rs62387743, rs62387744, rs62387746,rs62387747, rs62387748, rs62387749, rs62387750, rs62387751, rs62387972,rs62387973, rs62387975, rs62387976, rs62387986, rs62387987, rs62387988,rs62387993, rs35998688, rs36003434, rs62388008, rs62388009, rs62388010,rs62388011, rs36054439, rs36054502, rs36070865, rs36087347, rs36090082,rs36093776, rs36093807, rs36119103, rs36120552, rs41267702, rs41267704,rs45496399, rs55662629, rs55684678, rs62389046, rs62389047, rs62389048,rs58567314, rs58614013, rs58641936, rs58665157, rs58701160, rs58745226,rs58747981, rs58758067, rs58784041, rs58786258, rs58798370, rs58862118,rs58868196, rs58929118, rs58936812, rs58946642, rs58952738, rs59013552,rs59044073, rs59118746, rs59137061, rs59157013, rs59158709, rs59176676,rs59225235, rs59237300, rs59252024, rs59253371, rs59302766, rs59310777,rs59339642, rs59358244, rs59375420, rs59406771, rs59430691, rs59459480,rs59466516, rs59470271, rs59478891, rs59530583, rs59536682, rs59541156,rs59543599, rs59545339, rs59572963, rs59576590, rs59660553, rs59674857,rs59683312, rs59697453, rs59706950, rs59728940, rs59737568, rs59747547,rs59755079, rs59758474, rs62388012, rs62388013, rs62388972, rs62388974,rs62388975, rs62388976, rs62388977, rs62388978, rs62388979, rs62388980,rs62388981, rs62388982, rs62388983, rs62388984, rs62389001, rs62389002,rs62389003, rs62389004, rs62389005, rs62389033, rs62389034, rs62389035,rs62389036, rs62389037, rs62389038, rs62389039, rs62389040, rs62389041,rs62389042, rs62389043, rs72823523, rs72823524, rs63569360, rs63589760,rs66518516, rs66537562, rs66603893, rs66620531, rs66686151, rs66732320,rs66768766, rs66788546, rs66826504, rs66843340, rs66904455, rs67112370,rs67144687, rs67153659, rs67169180, rs67194841, rs67252205, rs67252206,rs67306835, rs67341869, rs67363532, rs67444717, rs67522722, rs67611932,rs67627414, rs67700003, rs67710364, rs75453237, rs67805820, rs67811348,rs67818802, rs67943406, rs68096947, rs68174733, rs70999323, rs70999325,rs70999327, rs70999329, rs70999330, rs70999333, rs70999334, rs70999336,rs70999338, rs70999339, rs70999340, rs70999343, rs71461894, rs71533660,rs71535078, rs71535082, rs71535083, rs71535084, rs71554566, rs71554567,rs71554568, rs71554569, rs71554570, rs71554571, rs71554572, rs71554573,rs71554574, rs71554575, rs71554576, rs71554577, rs71554578, rs71554579,rs71554580, rs71554581, rs71554582, rs71554583, rs71554584, rs71554585,rs71554586, rs71554587, rs71554588, rs71656942, rs71658367, rs71659990,rs71666629, rs71669096, rs71672303, rs71677734, rs71683630, rs71706080,rs71706411, rs71707826, rs71714432, rs71717905, rs71745682, rs71769107,rs71795785, rs71821274, rs71832745, rs71876194, rs71965340, rs71970468,rs72044303, rs72118488, rs72380402, rs72456659, rs72469012, rs72469947,rs72500881, rs72546604, rs72546605, rs72602306, rs72631315, rs72653742,rs72823506, rs72823507, rs72823510, rs72823512, rs72823517, rs72823525,rs72823529, rs72823597, rs72825516, rs72825518, rs72825519, rs72825521,rs72825523, rs72825525, rs72825527, rs72825529, rs72825531, rs72825533,rs72825535, rs72825541, rs72825544, rs72825545, rs72825549, rs72825552,rs72825554, rs72825555, rs72825556, rs72825558, rs72825562, rs72825565,rs72825568, rs72825569, rs72825570, rs72825571, rs72825572, rs72825573,rs72825574, rs72825575, rs72825578, rs72825585, rs72825588, rs72825589,rs72825594, rs72825597, rs72825601, rs72827809, rs72827822, rs72827827,rs72827833, rs72827835, rs72827836, rs72827837, rs72840230, rs72840232,rs72840234, rs72840239, rs72840240, rs72840241, rs72840244, rs72840246,rs72840247, rs72840248, rs73362674, rs73364707, rs73364709, rs73364711,rs73364751, rs73364776, rs73364785, rs73364798, rs73366705, rs73366713,rs73366721, rs73366748, rs73366761, rs73366765, rs73366771, rs73366776,rs73366778, rs73366779, rs73366798, rs73366801, rs73367622, rs73367623,rs73367633, rs73367661, rs73367672, rs73367677, rs73367680, rs73367686,rs73367694, rs73368708, rs73368709, rs73368722, rs73368723, rs73368728,rs73368730, rs73368732, rs73368754, rs73368758, rs73368759, rs73368772,rs73370208, rs73370288, rs73370659, rs73370662, rs73370663, rs73370667,rs73370671, rs73370673, rs73724831, rs73724832, rs73724833, rs73724835,rs73724836, rs73724837, rs73724839, rs73724840, rs73724841, rs73724842,rs73724843, rs73724845, rs73724847, rs73724848, rs73724850, rs73724857,rs73724858, rs73724859, rs73724861, rs73724863, rs73724866, rs73724867,rs73724868, rs73724871, rs73724872, rs73724873, rs73724875, rs73724876,rs73724877, rs73724879, rs73724880, rs73724881, rs73724882, rs73724883,rs73724884, rs73724885, rs73724886, rs73724887, rs73724888, rs73724889,rs73724890, rs73724891, rs73724893, rs73724894, rs73724895, rs73724896,rs73724897, rs73724899, rs73724900, rs73725205, rs73725207, rs73725208,rs73725209, rs73725212, rs73725213, rs73725214, rs73725217, rs73725218,rs73725219, rs73725220, rs73725221, rs73725222, rs73725223, rs73725224,rs73725225, rs73725227, rs73725228, rs73725229, rs73728008, rs73728010,rs73728013, rs73728015, rs73728016, rs73728018, rs73728019, rs73728020,rs73728021, rs73728022, rs73728024, rs73728025, rs73728026, rs73728031,rs74194628, rs74270213, rs74291718, rs74291719, rs74291733, rs74322237,rs74329810, rs74331294, rs74347472, rs74350904, rs74353119, rs74403517,rs74414010, rs74435795, rs74457420, rs75116343, rs75144496, rs75146463,rs75152280, rs75169721, rs75177708, rs75196773, rs75206772, rs75214939,rs75261311, rs75262123, rs75263959, rs75265046, rs75289325, rs75312646,rs75331189, rs75343357, rs75353806, rs75363259, rs75377995, rs75378259,rs75381541, rs75384244, rs75396966, rs75406553, rs75414284, rs75425053,rs75457709, rs75466802, rs75492142, rs75517189, rs75549413, rs75557885,rs75570360, rs75575322, rs75596063, rs75596846, rs75610748, rs75615925,rs75620575, rs75643921, rs75654158, rs75661547, rs75667456, rs75670595,rs75676584, rs75696115, rs75701256, rs75708790, rs75709295, rs75727213,rs75738583, rs75745599, rs72823520, rs72823522, rs76390822, rs76394540,rs76411435, rs76411875, rs76433420, rs76437621, rs76445886, rs76463104,rs76483804, rs76484837, rs76494113, rs76500024, rs76501379, rs76513322,rs76523673, rs76525317, rs76554075, rs76574906, rs76581598, rs76582567,rs76595137, rs76596897, rs76617903, rs76625882, rs76626055, rs76629559,rs76634972, rs76654064, rs76663417, rs76668407, rs76670120, rs76672287,rs76673595, rs76679902, rs76683358, rs76694802, rs76717034, rs76760544,rs76764881, rs76773302, rs76775876, rs76785860, rs76791107, rs76793969,rs76805420, rs76853695, rs76859260, rs76863558, rs76863681, rs76893457,rs76907939, rs76912480, rs76959295, rs76965950, rs76973173, rs76987576,rs77002402, rs77023672, rs77039792, rs77048757, rs77054452, rs77055558,rs77062811, rs77067927, rs77071183, rs77071433, rs77076128, rs77077211,rs77088625, rs77089026, rs77100378, rs77122408, rs77130504, rs77134018,rs77160486, rs77184786, rs77201521, rs77204851, rs77221523, rs77221793,rs77226339, rs77232892, rs77236307, rs77237180, rs77249707, rs77254763,rs77271510, rs77278511, rs77285430, rs77295905, rs77297675, rs77305520,rs77306065, rs77329045, rs77343049, rs77359482, rs77366702, rs77380837,rs77385838, rs77394211, rs77396486, rs77396677, rs77405592, rs77421598,rs77426312, rs77427088, rs77430100, rs77431308, rs77431610, rs77433209,rs77437711, rs77444599, rs77454273, rs77457072, rs77458480, rs77458661,rs77461267, rs77466572, rs77493467, rs77493561, rs77500907, rs77507217,rs77510342, rs77523729, rs77526291, rs77527285, rs77581643, rs77585879,rs77585905, rs77587606, rs77593545, rs77594463, rs77623816, rs77651884,rs77660263, rs77673709, rs77685186, rs77689066, rs77702208, rs77724881,rs77728344, rs77733482, rs77758642, rs77763911, rs77765820, rs77783756,rs77787102, rs77812167, rs77837566, rs77850573, rs77850645, rs77855830,rs77856665, rs77858497, rs77861691, rs77861873, rs77863051, rs77882646,rs77891423, rs77898026, rs77898059, rs77901801, rs77913923, rs77916634,rs74462581, rs74472288, rs74481682, rs74488074, rs74488081, rs74541363,rs74544103, rs74551385, rs74553632, rs74568782, rs74569248, rs74580155,rs74587439, rs74588175, rs74602985, rs74612133, rs74633735, rs74643077,rs74647203, rs74661942, rs74674985, rs74676523, rs74677012, rs74699452,rs74705806, rs74716584, rs74725436, rs74729778, rs74731559, rs74734737,rs74737314, rs74744439, rs74749190, rs74784855, rs74791754, rs74792074,rs74793513, rs74800515, rs74803189, rs74806026, rs74806030, rs74807394,rs74813727, rs74814820, rs74817128, rs74822921, rs74839173, rs74850737,rs74860524, rs74864976, rs74870209, rs74874352, rs74884853, rs74895193,rs74914370, rs74914706, rs74929720, rs74930178, rs74931603, rs74938038,rs74940560, rs74944537, rs74945243, rs74951974, rs74999854, rs75002482,rs75011557, rs75031674, rs75046313, rs75068405, rs75076424, rs75079392,rs75082618, rs75082763, rs75089449, rs75105269, rs75111702, rs75114260,rs75763713, rs75764758, rs75776513, rs75780040, rs75788456, rs75801396,rs75826989, rs75831937, rs75835015, rs75837702, rs75852370, rs75854945,rs75863848, rs75873938, rs75881756, rs75887878, rs75896125, rs75903292,rs75903535, rs75905425, rs75917858, rs75927437, rs75929627, rs75947436,rs75961462, rs75982803, rs76003317, rs76004137, rs76037091, rs76063370,rs76066134, rs76085695, rs76094202, rs76095931, rs76112039, rs76120093,rs76130767, rs76139833, rs76150204, rs76152292, rs76152304, rs76163792,rs76165370, rs76167482, rs76177153, rs76188893, rs76190976, rs76214718,rs76222135, rs76230878, rs76231931, rs76246942, rs76254550, rs76280058,rs76282370, rs76315178, rs76317739, rs76337230, rs76344407, rs76344901,rs76360736, rs76363631, rs76367447, rs76383129, rs76384465, rs78904757,rs78922334, rs78934255, rs78944473, rs78948490, rs78951523, rs78968436,rs79006576, rs79006912, rs79017917, rs79020982, rs79023029, rs79036696,rs79054583, rs79072094, rs79077000, rs79084729, rs79107575, rs79177140,rs79177217, rs79181244, rs79188747, rs79190262, rs79191370, rs79193989,rs79202621, rs79210016, rs79242487, rs79245462, rs79254575, rs79257409,rs79262688, rs79280618, rs79285842, rs79287368, rs79287686, rs79303516,rs79307303, rs79318826, rs79341431, rs79353347, rs79368864, rs79369029,rs79374471, rs79390898, rs79391472, rs79407069, rs79412324, rs79419034,rs77916869, rs77925603, rs77929060, rs77959611, rs77980141, rs78001098,rs78006815, rs78007550, rs78042397, rs78064404, rs78072812, rs78075485,rs78085193, rs78085685, rs78108220, rs78115249, rs78133384, rs78162517,rs78162972, rs78168954, rs78182473, rs78231134, rs78231489, rs78235172,rs78243253, rs78255781, rs78258857, rs78260927, rs78264267, rs78265670,rs78284375, rs78298418, rs78307011, rs78309184, rs78322985, rs78330169,rs78334148, rs78341114, rs78342056, rs78342281, rs78375753, rs78377677,rs78385232, rs78398193, rs78399351, rs78415485, rs78424976, rs78443260,rs78454991, rs78460474, rs78468017, rs78487690, rs78509571, rs78535369,rs78535800, rs78566245, rs78569047, rs78572828, rs78573290, rs78613242,rs78629508, rs78655546, rs78680685, rs78684855, rs78697934, rs78699134,rs78705466, rs78706477, rs78711673, rs78729178, rs78735808, rs78742531,rs78742717, rs78743834, rs78772107, rs78772864, rs78774412, rs78774554,rs78794721, rs78795878, rs78812434, rs78814376, rs78818147, rs78839772,rs78842859, rs78844305, rs78856205, rs78861401, rs78865350, rs78891031,rs78895862, rs78900557, rs78904100, rs112416730, rs113757490,rs112423127, rs112448047, rs112463780, rs112477802, rs112478697,rs112481189, rs112488299, rs112495707, rs112512714, rs112516254,rs112518023, rs112522439, rs112545191, rs112550124, rs112568386,rs112571096, rs112583508, rs112589477, rs112593783, rs112595095,rs112622104, rs112627471, rs112630364, rs112631519, rs112632928,rs112635253, rs112638703, rs112649636, rs112650001, rs112653142,rs112712942, rs112714435, rs112729878, rs112739217, rs112743537,rs112759347, rs112765917, rs112766264, rs112790112, rs112807462,rs112824481, rs112826413, rs112830463, rs112839209, rs112853609,rs112854777, rs112855934, rs112861371, rs79423133, rs79438489,rs79443962, rs79456110, rs79465246, rs79465944, rs79480639, rs79487465,rs79488288, rs79504485, rs79515017, rs79537203, rs79542015, rs79545411,rs79548991, rs79555374, rs79559656, rs79567385, rs79604916, rs79606367,rs79614556, rs79614670, rs79621451, rs79633732, rs79678305, rs79680988,rs79695628, rs79697898, rs79723397, rs79733883, rs79748348, rs79749413,rs79760610, rs79774197, rs79777883, rs79778808, rs79794335, rs79800590,rs79802251, rs79809996, rs79814073, rs79821521, rs79826522, rs79845609,rs79865225, rs79865952, rs79881269, rs79881757, rs79890331, rs79890580,rs79914902, rs79916459, rs79943477, rs79944650, rs79946188, rs79963215,rs79974547, rs79988973, rs79994578, rs79996628, rs80002251, rs80006940,rs80036911, rs80085771, rs80092476, rs80093112, rs80097029, rs80106233,rs80107224, rs80107778, rs80115059, rs80119849, rs80133275, rs80134524,rs80152335, rs80157586, rs80161311, rs80162668, rs80165549, rs80196848,rs80205567, rs80225531, rs80228032, rs80234647, rs80236140, rs80248228,rs80251478, rs80273982, rs80280007, rs80281835, rs80310484, rs80317727,rs80330118, rs80336726, rs80344484, rs80346600, rs80347638, rs80347782,rs80351174, rs111209264, rs111228543, rs111230250, rs111237357,rs111241295, rs111252274, rs111253148, rs111275569, rs111285998,rs111287565, rs111289778, rs111293246, rs111294286, rs111298421,rs111299691, rs111310488, rs112872908, rs112876660, rs112885511,rs112899072, rs112904209, rs112921996, rs112942106, rs112951110,rs112959484, rs112988232, rs112994179, rs112997423, rs113000878,rs113000917, rs113006227, rs113006530, rs113014563, rs113016437,rs113021034, rs113031060, rs113043241, rs113043694, rs113049083,rs113063214, rs113067516, rs113069452, rs113084706, rs113088997,rs113089959, rs113115852, rs113121741, rs113127730, rs113130256,rs111311176, rs111313235, rs111319686, rs111327465, rs111331252,rs111335034, rs111343926, rs111353075, rs111355545, rs111356518,rs111363301, rs111366085, rs111374017, rs111386164, rs111388477,rs111390753, rs111400418, rs111405337, rs111408397, rs111414893,rs111417029, rs111426375, rs111432136, rs111432826, rs111434517,rs111451193, rs111451489, rs111459676, rs111476258, rs111479179,rs111485195, rs111495825, rs111508487, rs111515393, rs111516255,rs111517072, rs111519888, rs111535985, rs111558083, rs111562283,rs111577925, rs111583280, rs111585013, rs111597397, rs111626252,rs111632785, rs111633671, rs111644033, rs111658041, rs111661539,rs111666021, rs111670084, rs111677701, rs111680880, rs111683640,rs111695554, rs111699482, rs111712243, rs111735010, rs111747621,rs111748658, rs111750552, rs111773391, rs111777244, rs111797452,rs111799385, rs111801569, rs111807904, rs111813336, rs111814287,rs111829323, rs111830296, rs111831951, rs111860606, rs111873965,rs111875611, rs111875886, rs111884236, rs111890224, rs111899568,rs111919785, rs111925901, rs111926475, rs111928647, rs111931936,rs111940183, rs111947094, rs111951254, rs111964724, rs111969171,rs111986222, rs112026393, rs112042384, rs112042712, rs112047619,rs112053959, rs112060044, rs112061894, rs112065052, rs112068062,rs112071614, rs112079900, rs112093863, rs112101163, rs112106634,rs112115987, rs112126818, rs112130910, rs112137981, rs112146576,rs112155442, rs112167822, rs112172398, rs112175378, rs112175731,rs112188181, rs112189481, rs112201456, rs112201752, rs112203139,rs112217043, rs112232082, rs112246571, rs112260078, rs112263021,rs112288961, rs112291700, rs112300384, rs112322780, rs112325934,rs112326315, rs112334718, rs112342550, rs112344373, rs112351111,rs112355979, rs112359289, rs112369407, rs112372488, rs112384260,rs112392920, rs112395922, rs112406931, rs113759963, rs113770895,rs113772107, rs113786082, rs113786256, rs113794735, rs113807928,rs113812627, rs113816822, rs113828128, rs113831854, rs113839052,rs113842642, rs113871296, rs113893969, rs113894371, rs113903902,rs113909060, rs113921124, rs113935100, rs113941924, rs113944102,rs113956591, rs113979949, rs114002033, rs114003737, rs114020848,rs114024521, rs114026017, rs114027647, rs114034162, rs114038263,rs114039376, rs114073172, rs114073896, rs114077935, rs114085835,rs114111635, rs114112534, rs114116009, rs114119090, rs114131546,rs114156031, rs114156434, rs114159445, rs114160825, rs114161799,rs114177593, rs114184148, rs114190550, rs114208038, rs114215580,rs114219177, rs114228627, rs114230242, rs114231879, rs114234613,rs114237657, rs114238249, rs114240707, rs114255266, rs114263401,rs114264388, rs114268504, rs114274437, rs114279270, rs114283078,rs114289404, rs114305746, rs114308848, rs114317491, rs114332746,rs114337946, rs114339891, rs114344437, rs114349891, rs114369537,rs114378930, rs114380116, rs114384998, rs114394594, rs114399486,rs114399679, rs114401328, rs114410508, rs114413734, rs114414155,rs114416481, rs114419697, rs114425903, rs114433458, rs114466279,rs114468123, rs114474432, rs114480336, rs114501007, rs114512287,rs114513901, rs114520284, rs114529137, rs114536718, rs114569359,rs114585115, rs114592041, rs114598241, rs114601082, rs114601949,rs114602929, rs114604248, rs114607447, rs114634970, rs114636761,rs114638308, rs114643370, rs114648800, rs114654034, rs114655470,rs114664730, rs114677387, rs114678829, rs114692921, rs114693682,rs114697505, rs114704739, rs114710576, rs114715336, rs114718414,rs114728928, rs114760086, rs114763167, rs114797514, rs114799032,rs114799658, rs114800833, rs114818442, rs114845371, rs114854973,rs114861654, rs114901606, rs114904408, rs114908784, rs114916711,rs114920290, rs114922456, rs114932025, rs114937629, rs114938585,rs114940160, rs114944342, rs114956838, rs114963928, rs114974805,rs114985754, rs114995173, rs115003246, rs115011674, rs115019946,rs115020492, rs115021474, rs115022568, rs115029812, rs115031085,rs115031411, rs115045821, rs115058708, rs115059912, rs115077346,rs115092038, rs115103523, rs115131758, rs115141003, rs115142386,rs115148803, rs115153649, rs115156122, rs115157301, rs115163058,rs115172870, rs115199126, rs115206751, rs115208003, rs115209071,rs115209215, rs115210681, rs115211732, rs115222697, rs115231235,rs115248274, rs115272821, rs115292247, rs115296246, rs115299728,rs115316930, rs115323417, rs115327013, rs115328876, rs115330971,rs113130763, rs113134965, rs113145342, rs113149327, rs113154128,rs115340884, rs115374372, rs115384603, rs115389105, rs115396707,rs115402311, rs115411230, rs115435873, rs115444662, rs115448706,rs115458098, rs115470191, rs115473350, rs115495406, rs115504894,rs115508308, rs115515986, rs115523123, rs115526062, rs115527092,rs115555758, rs115569391, rs115570050, rs115586962, rs115602708,rs115613288, rs115620347, rs115626317, rs115645304, rs115648841,rs115654424, rs115665760, rs115667449, rs115672065, rs115683048,rs115683979, rs115697918, rs115725050, rs115730713, rs115749548,rs115751435, rs115757803, rs115775639, rs115800675, rs115803171,rs115811491, rs115818721, rs115842876, rs115848436, rs115889732,rs115913944, rs115929150, rs115929734, rs115937736, rs115941713,rs115943989, rs115954477, rs115956999, rs115965793, rs115973050,rs115980786, rs115986810, rs115987186, rs115988852, rs116004683,rs116016276, rs116018244, rs116022005, rs116022608, rs116024418,rs116024786, rs116026336, rs113158189, rs113176122, rs113193263,rs113194976, rs113199968, rs113229821, rs113232779, rs113236140,rs113248097, rs113261740, rs113277942, rs113303990, rs113308917,rs113314131, rs113324245, rs113335568, rs113345902, rs113346825,rs113347315, rs113357231, rs113365631, rs113402430, rs113415655,rs113427133, rs113428074, rs113429334, rs113439237, rs113440250,rs113460221, rs113460989, rs113476374, rs113483178, rs113485461,rs113492691, rs113548322, rs113552502, rs113553348, rs113593442,rs113594805, rs113599607, rs113602069, rs113616128, rs113619308,rs113625415, rs113639243, rs113641661, rs113652596, rs113656336,rs113667138, rs113688169, rs113692259, rs113702805, rs113704812,rs113754227, rs113755256, rs116622222, rs116668861, rs116675131,rs116676668, rs116679328, rs116681469, rs116690959, rs116692085,rs116716193, rs116727056, rs116730306, rs116735942, rs116744799,rs116747278, rs116752358, rs116756625, rs116769438, rs116777003,rs116784848, rs116785615, rs116788211, rs116827356, rs116827506,rs116827647, rs116836825, rs116845946, rs116849750, rs116855116,rs116890067, rs116923572, rs116928640, rs116934384, rs116965552,rs116969658, rs116977158, rs116998029, rs117042012, rs117044798,rs117051577, rs117083616, rs138241563, rs138242027, rs138242325,rs138249208, rs138252877, rs138257305, rs138261708, rs138261797,rs138265863, rs138271652, rs138272271, rs138285498, rs138288856,rs138290142, rs138294451, rs138298443, rs138299893, rs138303660,rs138305602, rs138309730, rs138316202, rs138335264, rs138348376,rs138349692, rs138367568, rs138370098, rs138374028, rs138379754,rs138387353, rs138390044, rs138396773, rs138412972, rs138413460,rs138420553, rs138431014, rs138435789, rs138444767, rs138454998,rs138456591, rs138472925, rs138486232, rs138488390, rs138491275,rs138497591, rs138500563, rs138537319, rs138537524, rs138537872,rs138540726, rs138543495, rs138573527, rs138577769, rs138578377,rs138594240, rs138602493, rs138615559, rs138616465, rs138617413,rs138621697, rs138622178, rs138625599, rs138632189, rs138634232,rs138656545, rs138672964, rs138674101, rs138681841, rs138692593,rs138692732, rs138698546, rs138702167, rs138725881, rs138733884,rs138742614, rs138752694, rs138767035, rs138767586, rs138770220,rs138771076, rs138779024, rs138787562, rs138796295, rs138803446,rs138803849, rs138806521, rs138814973, rs138830983, rs138833403,rs138843275, rs138846942, rs138859218, rs138863737, rs138865765,rs138869364, rs138898571, rs138899972, rs138911441, rs138915725,rs138921826, rs138921833, rs138923020, rs138927047, rs138936448,rs138939256, rs138940819, rs138950337, rs138955475, rs138964995,rs138973800, rs138983477, rs138990326, rs139001446, rs139019175,rs139027780, rs139030983, rs139037499, rs139043475, rs139046406,rs140699518, rs139048428, rs139050317, rs139057035, rs139061789,rs139076194, rs139083950, rs139089995, rs139091518, rs139096712,rs139100179, rs139108831, rs139113374, rs139124745, rs139134201,rs139148755, rs139150364, rs139154350, rs139166270, rs139175810,rs139176988, rs139186298, rs139186513, rs139192838, rs139195459,rs139206275, rs116041670, rs116054279, rs116064041, rs116079497,rs116083256, rs116091802, rs139214412, rs139217038, rs139225641,rs139228441, rs139240996, rs139244424, rs139245402, rs139250680,rs139253106, rs139269193, rs139273900, rs139274278, rs139274397,rs139281364, rs139287457, rs139294618, rs139299191, rs139322945,rs139341026, rs139341425, rs139344679, rs139348538, rs139349112,rs139352286, rs139362274, rs139363370, rs139371535, rs139381022,rs139394947, rs139396162, rs139418848, rs139418899, rs139424374,rs139427172, rs116095191, rs116103784, rs116110305, rs116120591,rs116128992, rs116144132, rs116147639, rs116148844, rs116165474,rs116198025, rs116198427, rs116198721, rs116200123, rs116212230,rs116241524, rs116251582, rs116254845, rs116268241, rs116269613,rs116293158, rs116295725, rs116310680, rs116325143, rs116335610,rs116336378, rs116338086, rs116346644, rs116348046, rs116355128,rs116358652, rs116359566, rs116362405, rs116385824, rs116402152,rs116412685, rs116421940, rs116422397, rs116422597, rs116425793,rs116426329, rs116434547, rs116443433, rs116443813, rs116457971,rs116458307, rs116458644, rs116475331, rs116490162, rs116492549,rs116496021, rs116498652, rs116508717, rs116525777, rs116527646,rs116540327, rs116550345, rs116550515, rs116566515, rs116575622,rs116577166, rs116580910, rs116582778, rs116589930, rs116597856,rs116599639, rs139570990, rs141629115, rs141639027, rs141639123,rs141653644, rs141657132, rs139579956, rs139581375, rs139581623,rs139589322, rs139589609, rs139592563, rs139597632, rs139611918,rs139612558, rs139621654, rs139637611, rs139646259, rs139650714,rs139651031, rs139667378, rs139669380, rs139671524, rs139671758,rs139673291, rs139675938, rs139679034, rs139681934, rs139691034,rs139693322, rs139694734, rs139706522, rs139711484, rs139714840,rs139727434, rs139728229, rs139728305, rs139734471, rs139756764,rs139758640, rs139770427, rs139788614, rs139790577, rs139790949,rs139797073, rs117100786, rs117142006, rs117150537, rs117194829,rs117229057, rs117271107, rs117288265, rs117314725, rs117350069,rs117368753, rs117428429, rs117449542, rs117472758, rs117481787,rs117488164, rs117493509, rs117531865, rs117568415, rs117582296,rs117667497, rs117671216, rs117675384, rs117768666, rs117775916,rs117777068, rs117805814, rs117816075, rs117848696, rs117887154,rs117911460, rs117940539, rs117944283, rs117976803, rs117981077,rs117982177, rs118017239, rs118029607, rs118046275, rs118108568,rs118118835, rs118172138, rs137855401, rs137862588, rs137876188,rs137877472, rs137877493, rs137882977, rs137886933, rs137887046,rs137887872, rs137899324, rs137915644, rs137922677, rs137924776,rs137927783, rs137930358, rs137936389, rs137948328, rs137949677,rs137954323, rs137957791, rs137966768, rs137984678, rs137988256,rs137994876, rs137996121, rs138012520, rs138022846, rs138027978,rs138038301, rs138062979, rs138068854, rs138080053, rs138081071,rs138095906, rs138101962, rs138105392, rs138110677, rs138142237,rs138143853, rs138148309, rs138151094, rs138151314, rs138165340,rs138168106, rs138179168, rs138183684, rs138210135, rs138218898,rs138225483, rs138233584, rs139812958, rs139814556, rs139833986,rs139853750, rs139854940, rs139864000, rs139864180, rs139868779,rs139883489, rs139888970, rs139889328, rs139890574, rs139905979,rs139912409, rs139918586, rs139918898, rs139919577, rs139933179,rs139939284, rs139944366, rs140152644, rs140153287, rs140157476,rs140160532, rs140172970, rs140183885, rs140184778, rs140190736,rs140192492, rs140199633, rs140215283, rs140227809, rs140230223,rs140234021, rs140234563, rs140237220, rs140240031, rs140247236,rs140257227, rs140269427, rs140270335, rs140274213, rs140276633,rs140294917, rs140310448, rs140331199, rs140363104, rs140366005,rs140370240, rs140375115, rs140382411, rs140383509, rs140388902,rs140396432, rs140405760, rs140407811, rs140421628, rs140432115,rs140440557, rs140445808, rs140470305, rs140481432, rs140481570,rs140484131, rs140498392, rs140507568, rs140509096, rs140511551,rs140516135, rs140516480, rs140522600, rs140522754, rs140524018,rs140529391, rs140541338, rs140552583, rs140573517, rs140573672,rs140574425, rs140577185, rs140581123, rs140588886, rs140588972,rs140595822, rs140598474, rs140602251, rs140602570, rs140604739,rs140641937, rs140648458, rs140657248, rs140673713, rs140684682,rs140688049, rs140690626, rs140704104, rs140706503, rs140709024,rs140715913, rs140716500, rs140720384, rs140727440, rs140731274,rs140731493, rs140736454, rs140739342, rs140753497, rs140762737,rs140768325, rs140776889, rs140778898, rs140792278, rs140797279,rs140808957, rs140810986, rs140813563, rs140819438, rs140835064,rs140868389, rs140869551, rs140872650, rs140872849, rs140874783,rs140878584, rs140892011, rs140910387, rs140932939, rs140939806,rs140965223, rs140971015, rs140993902, rs140997529, rs141009599,rs141014408, rs141014519, rs141022714, rs141031222, rs141038129,rs141047554, rs141047649, rs141053125, rs141056728, rs141056859,rs141058831, rs141064654, rs141071761, rs141080404, rs141093199,rs141094055, rs141098441, rs141099005, rs141109813, rs141109856,rs141110236, rs141112771, rs141115418, rs141138789, rs141142224,rs141144985, rs141163860, rs141170425, rs139431431, rs139431810,rs139432762, rs139466640, rs139468277, rs139477850, rs139492474,rs139496823, rs139499740, rs139503642, rs139505322, rs139515536,rs139517495, rs139523054, rs139528895, rs139529487, rs139535192,rs139538317, rs139550605, rs141176557, rs141181980, rs141186519,rs141188583, rs141192353, rs141197223, rs141206302, rs141210794,rs141211585, rs141212712, rs141218018, rs141220673, rs141222116,rs141236170, rs141240251, rs141254572, rs141260739, rs141262983,rs141265668, rs141274783, rs141282030, rs141283083, rs141290441,rs141296755, rs141307800, rs141314975, rs141316435, rs141321658,rs141327013, rs141329200, rs141334254, rs141341470, rs141367169,rs141372934, rs141382123, rs141382992, rs141391935, rs141399499,rs141408476, rs141408742, rs141417222, rs141421171, rs141461294,rs141465067, rs141492833, rs139554502, rs139570929, rs141664123,rs141671471, rs141673939, rs141675287, rs141684515, rs141690849,rs141695102, rs141695949, rs141697899, rs141701429, rs141701569,rs141708349, rs141714838, rs141716380, rs141718507, rs141727898,rs141728458, rs141738820, rs141742244, rs141745300, rs141746407,rs141756630, rs141758316, rs141758568, rs141763638, rs141771066,rs141774435, rs141779634, rs141785369, rs141814708, rs141826171,rs141828086, rs141829643, rs141834926, rs141836403, rs141838020,rs141841786, rs141848558, rs141862886, rs141864897, rs141865485,rs141870598, rs141880614, rs141882280, rs141886937, rs141892482,rs141916416, rs141931702, rs141931798, rs141940593, rs141947547,rs141983282, rs141984408, rs141999713, rs142009486, rs142023328,rs142025135, rs142026521, rs142031988, rs142035460, rs142040013,rs142047345, rs142165829, rs142176029, rs142176359, rs142195406,rs142233682, rs142236902, rs142241754, rs142245266, rs142248338,rs142276834, rs142278041, rs142284212, rs142284796, rs142289593,rs142311437, rs142318620, rs142339743, rs142345723, rs142346638,rs142349688, rs142351745, rs142355507, rs142363558, rs142364065,rs142368830, rs142373444, rs142377162, rs142381143, rs142382830,rs142387391, rs142388015, rs142402694, rs142404162, rs142411159,rs142411724, rs142413404, rs142413691, rs142421640, rs142423255,rs142436132, rs142442939, rs142451973, rs142465263, rs142472465,rs142474365, rs142477613, rs142481822, rs142497714, rs142507362,rs142510815, rs142514164, rs142525842, rs142529362, rs142533130,rs142541099, rs142543027, rs142568310, rs142580001, rs142595807,rs142606018, rs142609698, rs142615301, rs142615417, rs142623668,rs142644102, rs142669928, rs142673128, rs142673819, rs142676146,rs142687886, rs142695042, rs142698360, rs142701292, rs139954671,rs139959144, rs139959906, rs139969341, rs139999045, rs140000371,rs140001382, rs140002636, rs140002996, rs140007635, rs140009890,rs140042005, rs140045725, rs140048093, rs140053216, rs140054095,rs140055197, rs140069979, rs140087096, rs140090971, rs140090990,rs140121067, rs142707728, rs142711630, rs142718286, rs142729700,rs142748777, rs142758665, rs142767442, rs142770327, rs142771410,rs142783282, rs142800801, rs142815432, rs142820144, rs142837305,rs142839094, rs142850959, rs142855990, rs142857304, rs142859390,rs142860655, rs142862205, rs142863892, rs142874920, rs142876092,rs142876660, rs142882261, rs142882444, rs142886316, rs142895205,rs142898323, rs142901493, rs142910124, rs142912182, rs142919666,rs142922664, rs142931035, rs142934924, rs142937046, rs142941394,rs142950610, rs142955121, rs142958498, rs142962695, rs142969089,rs142973090, rs142974694, rs142975616, rs142981911, rs142996070,rs142999359, rs143006944, rs143026101, rs143027594, rs143040254,rs143054803, rs143066951, rs143076837, rs143085342, rs143093747,rs143094976, rs143099582, rs143101211, rs143102716, rs143110024,rs143114756, rs143117186, rs143125796, rs143135801, rs143138436,rs143143294, rs143144787, rs143146876, rs143147048, rs143154695,rs143157234, rs143157569, rs143162188, rs143170537, rs143175820,rs143178986, rs143203547, rs143204278, rs143207282, rs143209272,rs143220064, rs143232243, rs143237604, rs143243981, rs143245440,rs143247068, rs143252512, rs143253642, rs143254710, rs143256883,rs143257924, rs143263459, rs143272800, rs145825148, rs145834637,rs145845531, rs145853738, rs143284119, rs143302184, rs143313443,rs143332218, rs143333778, rs143337548, rs143366995, rs143370264,rs143370405, rs143383305, rs143386858, rs143399384, rs143399560,rs143414224, rs143415004, rs143418496, rs143421851, rs143439533,rs143456299, rs143457075, rs143458830, rs143473538, rs143476055,rs143485184, rs143487285, rs143490303, rs143511518, rs143518175,rs143523999, rs143527268, rs143544922, rs143560442, rs143563705,rs143568749, rs143574943, rs143580235, rs143584546, rs143588744,rs143593843, rs143601415, rs143606757, rs143622403, rs143624504,rs143625562, rs143631998, rs143639560, rs143639858, rs143648265,rs143653693, rs143656214, rs143662383, rs143663615, rs143667847,rs143670505, rs143670692, rs143679294, rs143680503, rs143691277,rs143694540, rs143703955, rs143725487, rs143732790, rs143736829,rs143742095, rs143744419, rs143747150, rs143751686, rs143753931,rs143758741, rs143782957, rs143785440, rs143786882, rs143793724,rs143793905, rs143795942, rs143813141, rs143814852, rs143821277,rs143822384, rs143825557, rs143826952, rs143841038, rs143847959,rs143852387, rs143853274, rs143857059, rs143862420, rs143865008,rs143871777, rs143877789, rs143879969, rs143895055, rs143904151,rs143908297, rs143913910, rs143931565, rs143955086, rs143959633,rs143971026, rs143971223, rs141424993, rs141439463, rs141443979,rs141458671, rs141507325, rs141519665, rs141527718, rs141545152,rs141556542, rs141560842, rs141567259, rs141580746, rs141585880,rs141592840, rs141599542, rs141599655, rs141602179, rs141616108,rs141618033, rs141621137, rs144057459, rs144076731, rs144096425,rs144105428, rs144109553, rs144120901, rs144125511, rs144144440,rs144146812, rs144149858, rs144166792, rs144187607, rs144195914,rs144220619, rs144231290, rs144237796, rs144241291, rs144260845,rs144269669, rs144269957, rs144270497, rs144274162, rs144276049,rs144282025, rs144292130, rs144305851, rs144307438, rs144307843,rs144311368, rs144317951, rs144318370, rs144324512, rs144326428,rs144444917, rs144445351, rs144456420, rs144460392, rs144460876,rs144462044, rs144480603, rs144500672, rs144502277, rs144511159,rs144512832, rs144513982, rs144518091, rs142052564, rs142053583,rs142058254, rs142059161, rs142063883, rs142076443, rs142078189,rs142082338, rs142092358, rs142093576, rs142102521, rs142121091,rs142125219, rs142146508, rs142160943, rs142162538, rs144533684,rs144535463, rs144556313, rs144562054, rs144565843, rs144566585,rs144576939, rs144583284, rs144584850, rs144596891, rs144605739,rs144613186, rs144638703, rs144648865, rs144649705, rs144649883,rs144664320, rs144676357, rs144682252, rs144693846, rs144702458,rs144708406, rs144712170, rs144713242, rs144714659, rs144724938,rs144733152, rs144753607, rs144759115, rs144760970, rs144770926,rs144774963, rs144775398, rs144791460, rs144795428, rs144802499,rs144805699, rs144810498, rs144815348, rs144825320, rs144833593,rs144834952, rs144849277, rs144861023, rs144862299, rs144867792,rs144869647, rs144880908, rs144884955, rs144893413, rs144904405,rs144904494, rs144904701, rs144906487, rs144916658, rs144922292,rs144936216, rs144942615, rs144943215, rs144945682, rs144957004,rs144958549, rs144962740, rs144981302, rs144989964, rs145000650,rs145016411, rs145017131, rs145017547, rs145024504, rs145028577,rs145031577, rs145041955, rs145047027, rs145050976, rs145075324,rs145077912, rs145082631, rs145094808, rs145094852, rs145095709,rs145108851, rs145123345, rs145136167, rs145143774, rs145151086,rs145160903, rs145161294, rs145163874, rs145164783, rs145169454,rs145174144, rs145182585, rs145189661, rs145193845, rs145198090,rs145202760, rs145207036, rs145211120, rs145213526, rs145221770,rs145247488, rs145255259, rs145266087, rs145268800, rs145278160,rs145283995, rs145284226, rs145287942, rs145287971, rs145303721,rs145305083, rs145312278, rs145315664, rs145322177, rs145335207,rs145339254, rs145343887, rs145343939, rs145350211, rs145359717,rs145391092, rs145392716, rs145396833, rs145402050, rs145402142,rs145407309, rs145409592, rs145411761, rs145429894, rs145448915,rs145457740, rs145477218, rs145486106, rs145487710, rs145489437,rs145490433, rs145491853, rs145492536, rs145500846, rs145501450,rs145507738, rs145510675, rs145516319, rs145522493, rs145524986,rs145535169, rs145538080, rs145540867, rs145541557, rs145546250,rs145549085, rs145561747, rs145569331, rs145572815, rs145575447,rs145580481, rs145582940, rs145592931, rs145593523, rs145594888,rs145598374, rs145605557, rs145613649, rs145622406, rs148217315,rs145637346, rs145637351, rs145647799, rs145671693, rs145677317,rs145681653, rs145682142, rs145687944, rs145691240, rs145699545,rs145702267, rs145702727, rs145706146, rs145718091, rs145721938,rs145726282, rs145738025, rs145739728, rs145745088, rs145763191,rs145790838, rs145802160, rs145805579, rs145806368, rs145813082,rs145816850, rs145864988, rs145866950, rs145874329, rs145876861,rs145877179, rs145881995, rs145892078, rs145898793, rs145901825,rs145912953, rs145913787, rs145923933, rs145935830, rs145945138,rs145946476, rs145954919, rs145955861, rs145977640, rs145983587,rs145990477, rs145990781, rs145996771, rs145997929, rs145998120,rs145999658, rs146008775, rs146009489, rs146010847, rs146024353,rs146042983, rs146044877, rs146045857, rs146049481, rs146049869,rs146054039, rs146064764, rs146077654, rs146083133, rs146092433,rs146094409, rs146111490, rs146119470, rs146120250, rs146150572,rs146161018, rs146165450, rs146172919, rs146175374, rs146177811,rs146183718, rs146188488, rs146201415, rs146310114, rs146329176,rs146333073, rs146340047, rs146342979, rs146352973, rs146353713,rs146357374, rs146362200, rs146364623, rs146373716, rs146374933,rs146375265, rs146388778, rs146393693, rs146406644, rs143983337,rs143993556, rs144015332, rs144015414, rs144019063, rs146573598,rs146578099, rs146580305, rs146592437, rs144031530, rs144031607,rs144033390, rs146596536, rs146596938, rs146608539, rs146632732,rs146643135, rs146653003, rs146659282, rs146664433, rs144034010,rs144034946, rs146686418, rs146701646, rs146711041, rs146711794,rs146713637, rs146716937, rs146728068, rs146753713, rs146777083,rs146780926, rs146783982, rs146789972, rs146791717, rs146800740,rs146803875, rs146805501, rs146814053, rs146816036, rs146817731,rs146823984, rs146823986, rs146831513, rs146833624, rs146838550,rs146849514, rs146849985, rs146853248, rs146861435, rs146868404,rs146872964, rs146890923, rs146891899, rs146893753, rs144343115,rs144346691, rs144350999, rs144359510, rs144363905, rs144368158,rs144370432, rs144371632, rs144382649, rs144388270, rs144410932,rs144411643, rs144420539, rs144426523, rs144432516, rs144438576,rs146900457, rs146924852, rs146927730, rs146930429, rs146934687,rs146937752, rs146953420, rs146956693, rs146958000, rs146959836,rs146963151, rs146971506, rs146975403, rs146975784, rs146985737,rs146994728, rs146999919, rs147005338, rs147017673, rs147029860,rs147030196, rs147033616, rs147039850, rs147040077, rs147043094,rs147045302, rs147055222, rs147059578, rs147062702, rs147065160,rs147072599, rs147092260, rs147096969, rs147121270, rs147124544,rs147149254, rs147151969, rs147161703, rs147161962, rs147165912,rs147165920, rs147169079, rs147174169, rs147178324, rs147183503,rs147193594, rs147198269, rs147200979, rs147207993, rs147220757,rs147243600, rs147243625, rs147255445, rs147272304, rs147274470,rs147280957, rs147283804, rs147294360, rs147303626, rs147304147,rs147305468, rs147313363, rs147332332, rs147344055, rs147350439,rs147353633, rs147359145, rs147361664, rs147365225, rs147373131,rs147388885, rs147396889, rs147401526, rs147409579, rs147409820,rs147410830, rs147419552, rs147420876, rs147434374, rs147436482,rs147442700, rs147538801, rs147550139, rs147564620, rs147571852,rs147577993, rs147586614, rs147600346, rs147600673, rs147609204,rs147613556, rs147633786, rs147638509, rs147643813, rs147659532,rs147670416, rs147703616, rs147704443, rs147706459, rs147706512,rs147713104, rs147714876, rs147715147, rs147718397, rs147722621,rs147727903, rs147731257, rs147733686, rs147735339, rs147743873,rs147747282, rs147754040, rs147762552, rs147772997, rs147774533,rs147804847, rs147807732, rs147807918, rs147819333, rs147820523,rs147835571, rs147838562, rs147838915, rs147840525, rs147852540,rs147859866, rs147863479, rs147863858, rs147867465, rs147893117,rs147893530, rs147900680, rs147914484, rs147923809, rs147939563,rs147943602, rs147946010, rs147952508, rs147953763, rs147956994,rs147960525, rs147969060, rs147976015, rs147993122, rs147996175,rs148005418, rs148005463, rs148013127, rs148015664, rs148016979,rs148020491, rs148022692, rs148028261, rs148037173, rs148037711,rs148045068, rs148057704, rs148059053, rs148065439, rs148071858,rs148075273, rs148078711, rs148080439, rs148082548, rs148088236,rs148090020, rs148114246, rs148132809, rs148135039, rs148136250,rs148138835, rs148143063, rs148156215, rs148171610, rs148173611,rs148196135, rs148203946, rs148208344, rs146204415, rs146215551,rs146232527, rs146242089, rs146248378, rs146256378, rs146265734,rs146281479, rs146284575, rs146289669, rs146298192, rs146378566,rs146407742, rs146416592, rs146423233, rs146427563, rs146432662,rs146433968, rs146436679, rs146442502, rs146451059, rs146477448,rs146478581, rs146487784, rs146489625, rs146492132, rs146531341,rs146540203, rs146550970, rs146557770, rs146558017, rs146561790,rs146569084, rs146672404, rs146674698, rs146675071, rs146675673,rs146679289, rs146680261, rs146683206, rs146683268, rs149047518,rs150957427, rs149050173, rs149055178, rs149056683, rs149063862,rs149065803, rs149066370, rs149071714, rs149074349, rs149079473,rs149084979, rs149109693, rs149111829, rs149112959, rs149119774,rs149121663, rs149127222, rs149127720, rs149132704, rs149136994,rs149140098, rs149149426, rs149151396, rs149163233, rs149179766,rs149185140, rs149209334, rs149210946, rs149219284, rs149228045,rs149233199, rs149234761, rs149235283, rs149241908, rs149243816,rs149246876, rs149255942, rs149270786, rs149274017, rs149324933,rs149330723, rs149335995, rs149351987, rs149355960, rs149368017,rs149370345, rs149372504, rs151291156, rs149376309, rs149378735,rs149380118, rs149385730, rs149389827, rs149401023, rs149404532,rs149406170, rs149406470, rs149412562, rs149415494, rs149417426,rs149418089, rs149418303, rs149422924, rs149425853, rs149427563,rs149454633, rs149455867, rs149461255, rs149470649, rs149478429,rs149499794, rs149520196, rs149539379, rs149545849, rs149564996,rs149568650, rs149578359, rs149582172, rs147447612, rs147449173,rs147464264, rs147467180, rs147469259, rs147476956, rs147477726,rs147490733, rs147504248, rs147510485, rs147511029, rs147513868,rs147521378, rs147523901, rs147536377, rs150423599, rs150432194,rs150441496, rs150442635, rs150456477, rs150460561, rs150466207,rs150470423, rs150474730, rs150477501, rs150511968, rs150529499,rs150536388, rs150538806, rs150546489, rs150549450, rs150553949,rs150562725, rs150565574, rs150568195, rs150571347, rs150586513,rs150587535, rs150598924, rs150607950, rs150613381, rs150617543,rs150623417, rs150641978, rs150649488, rs148218816, rs148220421,rs148224743, rs148227566, rs148231342, rs148232779, rs148234033,rs148241640, rs148247220, rs148251394, rs148257750, rs148262162,rs148269687, rs148286558, rs148294029, rs148295040, rs148297620,rs148345549, rs148381084, rs148382139, rs148388307, rs148392826,rs148393661, rs148398329, rs148404083, rs148414448, rs148432883,rs148447015, rs148475055, rs148485251, rs148486566, rs148487913,rs148490141, rs148496016, rs148525226, rs148526921, rs148527850,rs148530094, rs148535471, rs148540860, rs148545258, rs148551668,rs148557202, rs148561205, rs148574095, rs148580499, rs148584639,rs148588553, rs148597249, rs148603582, rs148604945, rs148611734,rs148622108, rs148642284, rs148645832, rs148654581, rs148660683,rs148689787, rs148694099, rs148695839, rs148700579, rs148708147,rs148719904, rs148725857, rs148727527, rs148730939, rs148734291,rs148736258, rs148738520, rs148742548, rs148743788, rs148751612,rs148754460, rs148760115, rs181585459, rs148763210, rs148765123,rs148765753, rs148770548, rs148773246, rs150664391, rs150692969,rs150694018, rs150697745, rs150703935, rs150705181, rs150726509,rs150735681, rs150737473, rs150741738, rs150751950, rs148775626,rs148778351, rs148779805, rs148787869, rs148790216, rs148796801,rs148814604, rs148830518, rs148841420, rs148842043, rs148848787,rs148851107, rs148857865, rs150756146, rs148858676, rs148876447,rs148879593, rs150764426, rs150773399, rs150774992, rs150778623,rs150781040, rs150787590, rs150789955, rs150793945, rs150796082,rs150816258, rs150817572, rs150818516, rs150825968, rs150829314,rs150831436, rs150852584, rs150870362, rs150872637, rs150877863,rs150887341, rs150902799, rs148879974, rs148882620, rs148887404,rs148894756, rs148898337, rs148900352, rs148903266, rs148916105,rs148918537, rs148924655, rs148928599, rs148936705, rs148940480,rs148948962, rs148952167, rs148970654, rs148973712, rs148975910,rs149009167, rs149013040, rs150905121, rs149016130, rs149036138,rs150963148, rs150974327, rs150977786, rs150981810, rs151014746,rs151018532, rs151024431, rs151036684, rs151042715, rs151046402,rs151050395, rs151061405, rs151062682, rs151067716, rs151071234,rs151073552, rs151076633, rs151080741, rs151091390, rs151094480,rs151098965, rs151104603, rs151106325, rs151108294, rs151115582,rs151134478, rs151134523, rs151136530, rs151138912, rs151139197,rs151139733, rs151142048, rs151149012, rs151174162, rs151190868,rs151192658, rs151215786, rs151229715, rs151231976, rs151243361,rs151260593, rs151262838, rs151268207, rs151272055, rs151278109,rs151280648, rs151301085, rs151309871, rs151313854, rs151324325,rs151325754, rs151329818, rs151338958, rs180674864, rs180676298,rs180677811, rs180689214, rs180690322, rs180697432, rs180701804,rs180706369, rs180706997, rs180709683, rs180710382, rs180724784,rs180726968, rs180774896, rs180800427, rs180806966, rs180820944,rs180826959, rs180831977, rs180839829, rs180841092, rs180843926,rs180846987, rs180854506, rs149584881, rs149590403, rs149592228,rs149596472, rs149598108, rs149604792, rs149604900, rs149616307,rs149618496, rs149621749, rs149626609, rs149633930, rs149636473,rs149639008, rs149649375, rs149669904, rs149675731, rs149685469,rs149685663, rs149693630, rs149702319, rs149711610, rs149712678,rs149716995, rs149722773, rs149724863, rs149735716, rs149741315,rs149741696, rs149751784, rs149759428, rs149760827, rs149761121,rs149771799, rs149778805, rs149790055, rs149809577, rs149818993,rs149822883, rs149854136, rs149856843, rs149862735, rs149883811,rs149891742, rs149896094, rs149901340, rs149905394, rs149919465,rs149935700, rs149939494, rs149949627, rs149957220, rs149960703,rs180855854, rs180858059, rs180869174, rs180871737, rs180876645,rs180876777, rs180890934, rs180904454, rs180910859, rs180914642,rs180917011, rs180918759, rs180921638, rs180923507, rs180925494,rs149962806, rs149967337, rs149970585, rs149971277, rs149976761,rs149982092, rs149988131, rs149992044, rs149993107, rs149994525,rs149995087, rs150013760, rs150019337, rs150033148, rs150035288,rs150050637, rs150063216, rs150068668, rs150070731, rs150080409,rs150086196, rs150092675, rs150096770, rs150126228, rs150135688,rs150138782, rs150153668, rs150161978, rs150169396, rs150178191,rs150179986, rs150198355, rs150198749, rs150208444, rs150211483,rs150213780, rs150220087, rs150221012, rs150230683, rs150234341,rs150236839, rs150249028, rs150252656, rs150253410, rs150257820,rs150259273, rs150264882, rs150266724, rs150285907, rs150288799,rs150292643, rs150295169, rs150302683, rs150309546, rs150320597,rs150329670, rs150344292, rs150346303, rs150375142, rs150375774,rs150379076, rs150379512, rs150387851, rs150406086, rs150415967,rs150419188, rs150423257, rs180926823, rs180956685, rs180959032,rs180960754, rs180963290, rs180968855, rs180975839, rs180976232,rs180980922, rs180984994, rs180994017, rs180996527, rs181000193,rs181001794, rs181006001, rs181028436, rs181034063, rs181055823,rs181060761, rs181066887, rs181077110, rs181082385, rs181093184,rs181095857, rs181105840, rs181124186, rs181126279, rs181130145,rs181137901, rs183689249, rs183700245, rs181138917, rs181140292,rs181143619, rs181143832, rs181144285, rs181152632, rs181155569,rs181157015, rs181158647, rs181162165, rs181164553, rs181164797,rs181169141, rs181176387, rs181181258, rs181187238, rs181190441,rs181193494, rs181198138, rs181206055, rs181221715, rs181222702,rs181242824, rs181252353, rs181254332, rs181257710, rs181280744,rs181290147, rs181301274, rs181310632, rs181318396, rs181324067,rs181330008, rs181334336, rs183822022, rs181340084, rs181347112,rs181350723, rs181353323, rs181366307, rs181386468, rs181405227,rs181407853, rs181419661, rs181425118, rs181425763, rs181433565,rs181439863, rs181441232, rs181447347, rs181456046, rs181459068,rs181462538, rs181463011, rs181465885, rs181469226, rs181469365,rs181471459, rs181476611, rs181478917, rs181480079, rs181496210,rs181502089, rs181504807, rs181506795, rs181507452, rs181539427,rs181545373, rs181548103, rs181548362, rs181553277, rs181564829,rs181572398, rs181576608, rs181582579, rs181587603, rs181593861,rs181601243, rs181603240, rs181608791, rs181612194, rs181619397,rs181620097, rs181626467, rs181629728, rs181633442, rs181736779,rs181741606, rs181766566, rs181771176, rs181773391, rs181774614,rs181782172, rs181792004, rs181793057, rs181799246, rs181819628,rs181845735, rs181851164, rs184240297, rs184245737, rs181854773,rs181862296, rs181869980, rs184285632, rs181876079, rs181883574,rs181894059, rs184317820, rs184326727, rs181897106, rs181903235,rs181905490, rs181907170, rs181908616, rs181913942, rs181916578,rs181954285, rs181960511, rs181968016, rs181970865, rs181976225,rs181983135, rs181991797, rs182001002, rs182002073, rs150907442,rs150921619, rs150923160, rs184431621, rs184449691, rs150927653,rs150935647, rs150936816, rs150945358, rs182066706, rs182074774,rs182076167, rs182077633, rs182107266, rs182108040, rs182142841,rs182171728, rs182175495, rs182186892, rs182187033, rs182197799,rs182198659, rs182200385, rs182215223, rs182232854, rs182237239,rs182252879, rs182254620, rs182260578, rs182260909, rs182264870,rs182265880, rs182269909, rs182276529, rs182293333, rs182294837,rs182298006, rs182312132, rs182315215, rs182317116, rs182321264,rs182327762, rs182329720, rs182332325, rs182334103, rs182335521,rs182335915, rs182338662, rs182339605, rs182341067, rs182397568,rs182403031, rs182408291, rs182412330, rs182413653, rs182415124,rs182419980, rs182427518, rs182428559, rs182469044, rs182472141,rs182478360, rs182529918, rs182533913, rs182547110, rs182550007,rs182555072, rs182558432, rs182560305, rs182564161, rs182577964,rs182578197, rs182580919, rs182589068, rs182595822, rs182598003,rs182599006, rs182600773, rs182607180, rs182609606, rs182613210,rs182614784, rs182615972, rs182617049, rs182617376, rs182618460,rs182619974, rs182622636, rs182624316, rs182660896, rs182664264,rs182683525, rs182688763, rs182713658, rs182746741, rs182766192,rs182767272, rs182780432, rs182782796, rs182787535, rs182788298,rs182807185, rs182814884, rs182821369, rs182821503, rs182821909,rs182828893, rs182829998, rs182833465, rs182835761, rs182837163,rs182837367, rs182849383, rs182849733, rs182852780, rs182854280,rs182857210, rs182858061, rs182859232, rs182863841, rs182863995,rs182868510, rs182868744, rs182877723, rs182880230, rs182882414,rs182884592, rs182887023, rs182887286, rs182890274, rs182903668,rs182913973, rs182918428, rs182925908, rs182931252, rs182970838,rs182979963, rs182987052, rs182993700, rs182997084, rs183007100,rs183011828, rs183017876, rs183025930, rs183039435, rs183040439,rs183043631, rs183043691, rs183054111, rs183061778, rs183077315,rs183105474, rs183105864, rs183107447, rs183115754, rs183115946,rs183126447, rs183129119, rs183140153, rs183150958, rs183158619,rs183161856, rs183167373, rs183169819, rs183172859, rs183173612,rs183174692, rs183175303, rs183180777, rs183182714, rs183185152,rs183188045, rs183189918, rs183190790, rs183191790, rs183198612,rs183203477, rs183208199, rs183209760, rs183253953, rs183258423,rs183260446, rs183264770, rs183268592, rs183272273, rs183276595,rs183284299, rs183295055, rs183298098, rs183310793, rs183320181,rs183323871, rs183357047, rs183373894, rs183376102, rs183384218,rs183386193, rs183390284, rs183407144, rs183410215, rs183420957,rs183423979, rs183426234, rs183426858, rs183429244, rs183430353,rs183433679, rs183435166, rs183437050, rs183437206, rs183439873,rs183442527, rs183443321, rs183445699, rs183446569, rs183449779,rs183451214, rs183451579, rs183456678, rs183463011, rs183470409,rs183472831, rs183474050, rs183474830, rs183478079, rs183489730,rs183505381, rs183558388, rs183560843, rs183561425, rs183587085,rs183588221, rs183592076, rs183599915, rs183601573, rs183604912,rs183613353, rs183614757, rs183625650, rs183628409, rs183633208,rs183636349, rs183642348, rs183665909, rs183670253, rs183671616,rs183675899, rs183682047, rs183686189, rs183704974, rs183711756,rs183716793, rs183717759, rs183718661, rs183721676, rs183723574,rs183725954, rs183729785, rs183731174, rs183733144, rs183742063,rs183748609, rs183753059, rs183762120, rs183780916, rs183782809,rs183785512, rs183785746, rs183790386, rs183791222, rs183792339,rs183794336, rs183797765, rs183801251, rs183801319, rs183804418,rs183805914, rs183806650, rs183809272, rs183809642, rs183812927,rs183813537, rs183821978, rs183826723, rs183840897, rs183848499,rs183860175, rs183863305, rs183868659, rs183868981, rs183869118,rs183873979, rs183875376, rs183879867, rs183882910, rs183884427,rs183907495, rs183914054, rs183931945, rs183932281, rs183932451,rs183934220, rs183944297, rs183945477, rs183950792, rs183993488,rs184001567, rs184002297, rs184005160, rs184016262, rs184032046,rs184037269, rs184043186, rs184065422, rs184074219, rs184076896,rs184084286, rs184084597, rs184095122, rs184096147, rs184098578,rs184105868, rs184107031, rs184109607, rs184110145, rs184113028,rs184113477, rs184114598, rs184115007, rs184117167, rs184120686,rs184124246, rs184139687, rs184143868, rs184149339, rs181637160,rs181638326, rs181640363, rs181642098, rs181642882, rs181648163,rs181652183, rs181654757, rs181656084, rs181671433, rs181674517,rs181680740, rs181685007, rs181687627, rs181699089, rs181709873,rs181722175, rs181724451, rs181727410, rs184176584, rs184200732,rs184202256, rs184204789, rs184211886, rs184212232, rs184216145,rs184216266, rs184224333, rs184231920, rs184233035, rs184236380,rs184249645, rs184268019, rs184272544, rs184288251, rs184291559,rs184308478, rs184327938, rs184332793, rs184338637, rs184339344,rs184339549, rs184346602, rs184349491, rs184351999, rs184357441,rs184369261, rs184375973, rs184377562, rs184377853, rs184392005,rs182003599, rs182005828, rs182029909, rs182033117, rs182034764,rs182043408, rs182044139, rs182046431, rs182046945, rs182048869,rs182050450, rs184423926, rs182054369, rs182063244, rs184495057,rs184495679, rs184499487, rs184503238, rs184511952, rs184518056,rs184521192, rs184521994, rs184523367, rs184524085, rs184528012,rs184531149, rs184532682, rs184558796, rs184577383, rs184592553,rs184598793, rs184601148, rs184617078, rs184622445, rs184622698,rs184631925, rs184634353, rs184634705, rs184640643, rs184641139,rs184644817, rs184645518, rs184650259, rs184651532, rs184654225,rs184659585, rs184665239, rs184689309, rs184690817, rs184701789,rs184719498, rs184719667, rs184727769, rs184738313, rs184745022,rs184745066, rs184747315, rs184750258, rs184757180, rs184768177,rs184773442, rs184776188, rs184785781, rs184799475, rs184805999,rs184812657, rs184837425, rs184841259, rs184846507, 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rs185789826,rs185790093, rs185805480, rs185807376, rs185816200, rs185821046,rs185821789, rs185822595, rs185825480, rs185847896, rs185849643,rs185864166, rs185866835, rs185873965, rs185877837, rs185897300,rs185907005, rs185908232, rs185914215, rs185914792, rs185932255,rs185936073, rs185942340, rs185950516, rs185951598, rs185964619,rs185966465, rs185968580, rs185996054, rs186001852, rs186015721,rs186018251, rs186021991, rs186026255, rs186037787, rs186046697,rs186047004, rs186057151, rs186059535, rs186066499, rs186076416,rs186077328, rs186078251, rs186079913, rs186080687, rs186081516,rs186086945, rs186089692, rs186092370, rs186103347, rs186109349,rs186116837, rs186122670, rs186129077, rs186149464, rs186150452,rs186162152, rs186162404, rs186164861, rs186182169, rs186185680,rs186188124, rs186197260, rs186199079, rs186199669, rs186212689,rs186220704, rs186229552, rs186232846, rs186243918, rs188849105,rs186244061, rs186256982, rs186258461, rs186264004, rs186275642,rs186281359, rs186286118, 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rs193213116, rs193218225, rs193241667,rs193250245, rs193252995, rs193264441, rs193281957, rs193287288,rs199705371, rs199711711, rs199716501, rs199744696, rs199748168,rs199754297, rs199755139, rs199756018, rs199777523, rs199780797,rs199783840, rs199787204, rs199793999, rs199795863, rs199796847,rs199800197, rs199801935, rs199857108, rs199883617, rs199888472,rs199890496, rs199895496, rs199905522, rs199918233, rs199918801,rs199934662, rs199939933, rs199968448, rs199970391, rs199971177,rs199981008, rs200014523, rs200033075, rs200037721, rs200039897,rs200048550, rs200055299, rs200059182, rs200061917, rs200062366,rs200066246, rs200067111, rs200067398, rs200069049, rs200071239,rs200072692, rs200073164, rs200076642, rs200077488, rs200088542,rs200088633, rs200105223, rs200109516, rs200111316, rs200111645,rs200117618, rs200117950, rs200137988, rs200138680, rs200142014,rs200159765, rs200160308, rs200163019, rs200163746, rs200183401,rs200190370, rs200194452, rs200199069, rs200202345, 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rs199511773,rs199527183, rs199546710, rs199552079, rs199557613, rs199558950,rs199578022, rs199591858, rs199595059, rs199637781, rs199653355,rs199654872, rs199655402, rs199662251, rs199667837, rs199669847,rs199678584, rs199679844, rs199691297, rs199696876, rs368045376,rs368052092, rs368060446, rs368062098, rs368070183, rs368074660,rs368076218, rs368078692, rs368086280, rs368094361, rs368102396,rs368108249, rs368108414, rs368108627, rs368109629, rs368115532,rs368126490, rs368135357, rs368136648, rs368137408, rs368139235,rs368153243, rs368153628, rs368154036, rs368166949, rs368187712,rs368191886, rs368217223, rs368218734, rs368218879, rs368231593,rs368233009, rs368238247, rs368240446, rs368251767, rs368267692,rs368270480, rs368274890, rs368297585, rs368299956, rs368316810,rs368327789, rs368338522, rs368343645, rs368346295, rs368365398,rs368367853, rs368368972, rs368381864, rs368399660, rs368402579,rs368408248, rs368408483, rs368419089, rs368421491, rs368427997,rs368432650, rs368440131, 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rs372174140, rs372177455, rs372200196,rs372206682, rs372219399, rs372220800, rs372224189, rs372226481,rs372227043, rs372241609, rs372251196, rs372252513, rs372253019,rs372271469, rs372280090, rs372285148, rs372295251, rs372295296,rs372318013, rs372319136, rs372319461, rs372335175, rs372349437,rs372349764, rs372358044, rs372385188, rs372385730, rs372387982,rs372399152, rs372403701, rs372406871, rs372407163, rs372416546,rs372417847, rs372425029, rs372431545, rs372436530, rs372436820,rs372447763, rs372449701, rs372451917, rs372464607, rs372476471,rs372479926, rs372480350, rs372491091, rs372491251, rs372494436,rs372495982, rs372509954, rs372519955, rs372533683, rs372534697,rs372542775, rs372557164, rs372593592, rs372599751, rs372606977,rs372635506, rs372642061, rs372644195, rs372649873, rs372650863,rs372651014, rs372674863, rs372685570, rs372686797, rs372692640,rs372709403, rs372737339, rs372751943, rs372753692, rs372770712,rs372772666, rs372773233, rs372786969, rs372788969, rs372797212,rs372798043, rs372799185, rs372803832, rs372804809, rs372841937,rs372845226, rs372854614, rs372867632, rs372891002, rs372892943,rs372901552, rs372903505, rs372927760, rs372928562, rs372962412,rs372968934, rs372975008, rs372982094, rs372986052, rs372999870,rs373015640, rs373021845, rs373025296, rs373028542, rs373036051,rs373039126, rs373039136, rs373047146, rs373055446, rs373062173,rs373073257, rs373076006, rs373076472, rs373077011, rs373089662,rs373108157, rs373122164, rs373138936, rs373144831, rs373146910,rs373156075, rs373172104, rs373176408, rs373177176, rs373179161,rs373184440, rs373191989, rs373195102, rs373206880, rs373229388,rs373236877, rs373240503, rs373242926, rs373246074, rs373249900,rs373266163, rs373267457, rs373276400, rs373288202, rs373289166,rs373307230, rs373307304, rs373309836, rs373313063, rs373315046,rs373336291, rs373340050, rs373346750, rs373349443, rs373363752,rs373374403, rs373376649, rs373386818, rs373394966, rs373412257,rs373423517, rs373427876, rs373443037, rs373457451, rs373491206,rs373492693, rs373495830, rs373504804, rs373507573, rs373508006,rs373511027, rs373516194, rs373523557, rs373527505, rs373557364,rs373561907, rs373568664, rs373573253, rs373576734, rs373577026,rs373578152, rs373585569, rs373590872, rs373594687, rs373611376,rs373632247, rs373650470, rs373673215, rs373698072, rs373726230,rs373726370, rs373726451, rs373730752, rs373736172, rs373736523,rs373751075, rs373752325, rs373775855, rs373781126, rs373790399,rs373795547, rs373809346, rs373813996, rs373815107, rs373816394,rs373817989, rs373820183, rs373825897, rs373830017, rs373830125,rs373832726, rs373833872, rs373835038, rs373835934, rs373841280,rs373847431, rs373848610, rs373851614, rs373853263, rs373856458,rs373873593, rs373883056, rs373886318, rs373892889, rs373894425,rs373904085, rs373908931, rs373917139, rs373922931, rs373923957,rs373927429, rs373930833, rs373939265, rs373949855, rs373951074,rs373959125, rs373965694, rs373966779, rs373968937, rs373975149,rs373981095, rs374000046, rs374010588, rs374014054, rs374019140,rs371328221, rs371330442, rs371330863, rs371330931, rs371336519,rs371339922, rs371344584, rs371351042, rs371382202, rs371382424,rs371389486, rs371403809, rs371420072, rs371430691, rs371437048,rs371443744, rs371448274, rs371454016, rs371457073, rs371461626,rs371469830, rs371470637, rs371477096, rs371503012, rs374028547,rs374034156, rs374034423, rs374037279, rs374038608, rs374057275,rs374057863, rs374065662, rs374081174, rs374092117, rs374094507,rs374099828, rs374106539, rs374140125, rs374149142, rs374170905,rs374178081, rs374192898, rs374203947, rs374218063, rs374222048,rs374224343, rs374234767, rs374237741, rs374243209, rs374247440,rs374249475, rs374264310, rs374275306, rs374290566, rs374292399,rs374299153, rs374310691, rs374322824, rs374335017, rs374338186,rs374341025, rs374348443, rs374349677, rs374358519, rs374363297,rs374381204, rs374383820, rs374383940, rs371522317, rs371528463,rs374421434, rs386459511, rs374427751, rs374439859, rs374463812,rs374464100, rs374495945, rs374524251, rs374524730, rs374526341,rs374530653, rs374531474, rs374532076, rs374532959, rs374534258,rs374541409, rs374543217, rs374570410, rs374577951, rs374586280,rs374588264, rs374593824, rs374594199, rs374610788, rs374613096,rs374618097, rs375359789, rs375360364, rs374391304, rs374404800,rs374408332, rs374414123, rs374416428, rs386697492, rs386697497,rs386697499, rs386697500, rs386697508, rs386697510, rs397689607,rs397692031, rs397692335, rs397713320, rs397717706, rs374618131,rs374634876, rs374637089, rs374640513, rs374654664, rs374667325,rs374667678, rs374677770, rs374679244, rs374684232, rs374684809,rs374688664, rs374698267, rs374699000, rs374699453, rs374723197,rs374767624, rs374776382, rs374782382, rs374782986, rs374797778,rs374803020, rs374811863, rs374820885, rs374824861, rs374838296,rs374838484, rs374850856, rs374852068, rs374856631, rs374859578,rs374881112, rs374901302, rs374911543, rs374924092, rs374924287,rs374935642, rs374953865, rs374960027, rs374964448, rs374966291,rs374966863, rs374969017, rs374971016, rs374973160, rs374979404,rs374982318, rs374989895, rs375008260, rs375014167, rs375028294,rs375030509, rs375071827, rs375073915, rs375087892, rs375126567,rs375162850, rs375177236, rs375189199, rs375190045, rs375201704,rs375206330, rs375226770, rs375259450, rs375264173, rs375264265,rs375264388, rs375269913, rs375277840, rs375287206, rs375304047,rs375309772, rs375326916, rs375328906, rs375331622, rs375356775,rs375383966, rs375409169, rs375412750, rs375413361, rs375416081,rs375416310, rs375420616, rs375433043, rs375438982, rs375439414,rs375466685, rs375472168, rs375481262, rs375488850, rs375508636,rs375510337, rs375525131, rs375528215, rs375531072, rs375540427,rs375550049, rs375550190, rs375550855, rs375568137, rs375604353,rs375614327, rs375637802, rs375639825, rs375643407, rs375643604,rs375666501, rs375671008, rs375675083, rs375685631, rs375693732,rs375694273, rs375696814, rs375700575, rs375701078, rs375710511,rs375715147, rs375719686, rs375729672, rs375733571, rs375735385,rs375739489, rs375745277, rs375748665, rs375757869, rs375767825,rs375791977, rs375794437, rs375797508, rs375805677, rs375809826,rs375814947, rs375824754, rs375841576, rs375845662, rs375853740,rs375858225, rs375891157, rs375894494, rs375900663, rs375912478,rs375912998, rs375930915, rs375941456, rs375944841, rs375949361,rs375954678, rs375959566, rs375970547, rs375983543, rs375985324,rs375988160, rs375989175, rs376005676, rs376006117, rs376010571,rs376010997, rs376018057, rs376037411, rs376071889, rs376080964,rs376084899, rs376092166, rs376107822, rs376127768, rs376137883,rs376142540, rs376163508, rs376171056, rs376180722, rs376191811,rs376195129, rs376213642, rs376224055, rs376232145, rs376233432,rs376236279, rs376247504, rs376266958, rs376274815, rs376278069,rs376285076, rs376288844, rs376289581, rs376290857, rs376297772,rs376304173, rs376308024, rs376312588, rs376317076, rs376321683,rs376326001, rs376326477, rs376326755, rs376329719, rs376330390,rs376344725, rs376349614, rs376371267, rs376387880, rs376402963,rs376406126, rs376413407, rs376420477, rs376423675, rs376426481,rs376427767, rs376432289, rs376439384, rs376455201, rs376475271,rs376482095, rs376487340, rs376488738, rs376502081, rs376526654,rs376528914, rs376532982, rs376547363, rs376555763, rs376560101,rs376562152, rs376573997, rs376576038, rs376581013, rs376582982,rs376584873, rs376599096, rs376608374, rs376608923, rs376610588,rs376626667, rs376656834, rs376665568, rs376665696, rs376675051,rs376685975, rs376687617, rs376689614, rs376694815, rs376705169,rs376706209, rs376723493, rs376739791, rs376744368, rs376750430,rs376775709, rs376788920, rs376791416, rs376797949, rs376805732,rs376815020, rs376817641, rs376823884, rs376823955, rs376826008,rs376832186, rs376833135, rs376835596, rs376854067, rs376857810,rs376862584, rs376871475, rs376873395, rs376886387, rs376897272,rs376898796, rs376921985, rs376940448, rs376944863, rs376955882,rs376978634, rs376988783, rs377004875, rs377009771, rs377021363,rs377048036, rs377050294, rs377058579, rs377059564, rs377074801,rs377087142, rs377095820, rs377098762, rs377102326, rs377123481,rs377133455, rs377140263, rs377145859, rs377147941, rs377148275,rs377166092, rs377177427, rs377181770, rs377195657, rs377210427,rs377255307, rs377259520, rs377260023, rs377266794, rs377284580,rs377286856, rs377292593, rs377336602, rs377343594, rs377356058,rs377370460, rs377372394, rs377384014, rs377387308, rs377393625,rs377394583, rs377404828, rs377408729, rs377433237, rs377433408,rs377437835, rs377443840, rs377467137, rs377469835, rs377496720,rs377498110, rs377503385, rs377503855, rs377521337, rs377522678,rs377528083, rs377536890, rs377557600, rs377557837, rs377559897,rs377565219, rs377565762, rs377565899, rs377571907, rs377572553,rs377577109, rs377583106, rs377584684, rs377585322, rs377592928,rs377598266, rs377600782, rs377603151, rs377610355, rs377610927,rs377646713, rs377662674, rs377662842, rs377664483, rs377665570,rs377686886, rs377696635, rs377715610, rs377719145, rs377720065,rs377721950, rs377722195, rs377722698, rs377735649, rs377736022,rs386406269, rs386406271, rs386406276, rs386406277, rs386406278,rs386406279, rs386406280, rs397940214, rs397949102, rs397949397,rs397950234, rs397952941, rs397961744, rs397962781, rs397965011,rs397970336, rs397972081, rs397975811, rs397718326, rs397720094,rs397720395, rs397723226, rs397744704, rs397746510, rs397754297,rs397770256, rs397773868, rs397776502, rs397791077, rs397792455,rs397809166, rs397814808, rs398000636, rs398000637, rs398000638,rs398000639, rs398000640, rs398000641, rs398000642, rs398000644,rs398000645, rs398000646, rs398000647, rs398000648, rs398000649,rs398000650, rs398048404, rs398065596, rs398065597, rs398065599,rs398084777, rs398084778, rs398084779, rs398084780, rs398094030,rs398094031, rs398094032, rs398094033, rs398109887, rs398109889,rs398109890, rs527237705, rs527241987, rs527372581, rs527380052,rs527382836, rs527388287, rs527395469, rs527405916, rs527410514,rs527410860, rs527416469, rs527428844, rs527433907, rs527443703,rs527454259, rs527458430, rs527463060, rs527464183, rs527464418,rs527492656, rs527499947, rs527522971, rs527524992, rs527525940,rs527532063, rs527537452, rs527540878, rs527541713, rs527549568,rs527551970, rs527552154, rs527554648, rs397833087, rs397841582,rs527559559, rs527562497, rs527568095, rs527573013, rs527575820,rs527596859, rs527597603, rs527608518, rs527614996, rs527626331,rs527633479, rs527636463, rs527638135, rs527643238, rs527649371,rs527652438, rs527656765, rs527668729, rs527676347, rs527677625,rs527686515, rs527686792, rs527687727, rs527698270, rs527699208,rs527706560, rs527713548, rs527715853, rs527724078, rs527725029,rs527752730, rs527755322, rs527771312, rs527773491, rs527785249,rs527790387, rs527792987, rs527799969, rs527806006, rs527810774,rs527811015, rs527812937, rs527813559, rs527826610, rs527832512,rs527838790, rs527844120, rs527860133, rs527863194, rs527878829,rs527878917, rs527904549, rs527904576, rs527917316, rs527922976,rs527923295, rs527924453, rs527927183, rs527933880, rs527939842,rs527950221, rs527955502, rs527956507, rs527974828, rs527976801,rs527979482, rs527983239, rs527991296, rs527991964, rs527993177,rs527995288, rs527998577, rs527999154, rs528000055, rs528006861,rs528006890, rs528015967, rs528019105, rs528023065, rs528039756,rs528047551, rs528052994, rs528063222, rs528066165, rs528073888,rs528083154, rs528085350, rs528091650, rs528094309, rs528097775,rs528102245, rs528109429, rs528115971, rs528118266, rs528125083,rs528132275, rs528140556, rs528150098, rs528158925, rs528167754,rs528175367, rs528210168, rs528232864, rs528232913, rs528234405,rs528245091, rs528255250, rs528262998, rs528268179, rs528271062,rs528272006, rs528278110, rs528278130, rs528295444, rs528297749,rs528307819, rs528308424, rs528316209, rs528329304, rs528330182,rs528340553, rs528344220, rs528355258, rs528359061, rs528369916,rs528381269, rs528401336, rs528401422, rs530697932, rs528402948,rs528407393, rs528409152, rs528411255, rs528436817, rs528437073,rs528443794, rs528444035, rs528449853, rs528451152, rs528452174,rs528453166, rs528456589, rs528461983, rs528466804, rs528467105,rs528477001, rs528478786, rs528482407, rs528482646, rs528485574,rs528487232, rs528490361, rs528498541, rs528498836, rs528506004,rs528518476, rs528519354, rs528519650, rs528528520, rs528533384,rs528535721, rs528539709, rs528539735, rs528541324, rs528541813,rs528543540, rs528547935, rs528562073, rs528585941, rs528587846,rs528599011, rs528599321, rs528603088, rs528613919, rs528614378,rs528622562, rs528630866, rs528645252, rs528651793, rs528653372,rs528661601, rs528663529, rs528664246, rs528674256, rs528677851,rs528689181, rs528709478, rs528710093, rs528715640, rs528717431,rs528718206, rs528722549, rs528724437, rs528737652, rs528747556,rs528750837, rs528760435, rs528761252, rs528770114, rs528774613,rs528795526, rs528805066, rs528809286, rs528819863, rs528826678,rs528830049, rs528832610, rs528834173, rs528839843, rs528840165,rs528858840, rs528863964, rs528869577, rs528871390, rs528874947,rs528876912, rs528883521, rs528884494, rs528900691, rs528905942,rs528917509, rs528921532, rs528931874, rs528945608, rs528951611,rs528985779, rs528987731, rs528988293, rs528993173, rs528996519,rs529003331, rs529008248, rs529010750, rs529012677, rs529014131,rs529018441, rs529028323, rs529032520, rs529039842, rs529051502,rs529065733, rs529078825, rs529080965, rs529098743, rs529106043,rs529115667, rs529119245, rs529134172, rs529134736, rs529134818,rs529141151, rs529144050, rs529144407, rs529149131, rs529151460,rs529152610, rs529153729, rs529154195, rs529165734, rs529171686,rs529175748, rs529178644, rs529192013, rs529209483, rs529229867,rs529232881, rs529256673, rs529257363, rs529263004, rs529269770,rs529273798, rs529277915, rs529283002, rs529287319, rs529294776,rs529301601, rs529310988, rs529311830, rs529316661, rs529320697,rs529323056, rs529325545, rs529327388, rs529339517, rs529344302,rs529344355, rs397841583, rs397841584, rs397841804, rs397885129,rs397934883, rs529389969, rs529403295, rs529405026, rs529406830,rs529407839, rs529420265, rs529443892, rs529449075, rs529466542,rs529475689, rs529479290, rs529482076, rs529484475, rs529489501,rs529494522, rs529494741, rs529503936, rs529505715, rs529511365,rs529517989, rs529518355, rs529519881, rs529531325, rs529534849,rs529537344, rs529542607, rs529559451, rs529560384, rs529564930,rs529566955, rs529568620, rs529574979, rs529576275, rs529580088,rs529587937, rs529607333, rs529620354, rs529621241, rs529621492,rs529636280, rs529636705, rs529643549, rs529650112, rs529653450,rs529653701, rs529670457, rs529671163, rs529681724, rs529684431,rs529688810, rs529690176, rs529693115, rs529694186, rs529704927,rs529707470, rs529707733, rs527255635, rs527273339, rs527274195,rs527277446, rs527286783, rs527286811, rs527287145, rs527320194,rs527322365, rs527331199, rs527336019, rs532067220, rs527350004,rs527357752, rs527363270, rs527372335, rs529714000, rs529725097,rs529741564, rs529752515, rs529754696, rs529758204, rs529762154,rs529769455, rs529784244, rs529798323, rs529803122, rs529808050,rs529816892, rs529819550, rs529832214, rs529840866, rs529843476,rs529851419, rs529853091, rs529860767, rs529863181, rs529877657,rs529878913, rs529883330, rs529885210, rs529886925, rs529889757,rs529890318, rs529894419, rs529896107, rs529909472, rs529929543,rs529936773, rs529951956, rs529955243, rs529963213, rs529978372,rs529979090, rs529986971, rs530009508, rs530019372, rs530028811,rs530039212, rs530040037, rs530043573, rs530058172, rs530059312,rs530060658, rs530063065, rs530065930, rs530067338, rs530068648,rs530069575, rs530070054, rs530074772, rs530076674, rs530078533,rs530086546, rs530094988, rs530123811, rs530124831, rs530378325,rs530382650, rs530459191, rs530461401, rs530464993, rs530473785,rs530476903, rs530478593, rs530486875, rs530497724, rs530499618,rs530513982, rs530521043, rs530523417, rs530531609, rs530538883,rs530543405, rs530543732, rs530546744, rs530553571, rs530560574,rs530561234, rs530561356, rs530567241, rs530574430, rs530580417,rs530582057, rs530583345, rs530583688, rs530588889, rs530593099,rs530601057, rs530604524, rs530606642, rs530607959, rs530609239,rs530618085, rs530627183, rs530630679, rs530632892, rs530644713,rs530647782, rs530647881, rs530654915, rs530658600, rs530664239,rs530676855, rs530683135, rs530697292, rs530700732, rs530707793,rs530712643, rs530722725, rs530727615, rs530728524, rs530728710,rs530737488, rs530751825, rs530755035, rs530756839, rs530766764,rs530771964, rs530776022, rs530779614, rs530788654, rs530794515,rs530797294, rs530820388, rs530828961, rs530829691, rs530836975,rs530838446, rs530853073, rs530854030, rs530858557, rs530861197,rs530870779, rs530880082, rs530883421, rs530883976, rs530885953,rs530888130, rs530900128, rs530904894, rs530913477, rs530914442,rs530915545, rs530922181, rs530926914, rs530940141, rs530956151,rs530962264, rs530968809, rs530977901, rs530985550, rs530995203,rs530996449, rs531007335, rs531014207, rs531020075, rs531027424,rs531031542, rs531037720, rs531044961, rs531045381, rs531057673,rs531066369, rs531068037, rs531072203, rs531086456, rs531091762,rs531093009, rs531104626, rs531107700, rs531111045, rs531119744,rs531130943, rs531152117, rs531154528, rs531157931, rs531162934,rs531163741, rs531167919, rs531169236, rs531171009, rs531172171,rs531173825, rs531179681, rs531180905, rs531193017, rs531197304,rs531198142, rs531200187, rs531200667, rs531202895, rs531205054,rs531205571, rs531212994, rs531215409, rs531220819, rs531221022,rs531226321, rs531227574, rs531231764, rs531234640, rs531236562,rs531243301, rs531255898, rs531258145, rs531260558, rs531268933,rs531288539, rs531300919, rs531314233, rs531314501, rs531330020,rs531332490, rs531339665, rs531340979, rs531345768, rs531362446,rs531371404, rs531385015, 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rs539150219, rs539155879, rs539224030,rs539238259, rs539245123, rs539246138, rs539247385, rs539255541,rs539259329, rs539263639, rs539264646, rs537045100, rs537059499,rs537062630, rs537077713, rs537078952, rs537086126, rs537089072,rs537089147, rs537102584, rs537104749, rs537107794, rs537112101,rs537114376, rs537116881, rs537120416, rs537129176, rs537147291,rs537154154, rs537154234, rs539277444, rs539283031, rs537167141,rs537188133, rs537193544, rs537195970, rs537205853, rs537207136,rs537246497, rs537248766, rs537249754, rs537252519, rs537256405,rs537261149, rs537261731, rs537271186, rs537280163, rs537300289,rs537317787, rs537318621, rs537321230, rs537322659, rs539314383,rs539318288, rs537342423, rs537358923, rs537361228, rs537368188,rs539319658, rs539344721, rs539351915, rs539354094, rs539360589,rs539363170, rs539367779, rs539389751, rs539390268, rs539404600,rs539419560, rs539432768, rs539442690, rs539445189, rs539449770,rs537372723, rs537386961, rs537397553, rs537397931, rs537410517,rs537411480, rs537414371, rs537425236, rs537431600, rs537432206,rs537438385, rs537438976, rs537439942, rs537452483, rs537456419,rs537459561, rs537469025, rs537470304, rs537471919, rs537472661,rs537472983, rs537477622, rs537489371, rs537497614, rs537500852,rs537507718, rs537508220, rs537516115, rs537516914, rs537524455,rs537532155, rs537537670, rs537544467, rs537546571, rs537553704,rs537560120, rs537561999, rs537562008, rs537574535, rs537578489,rs537585140, rs537585866, rs537592320, rs537597164, rs537601615,rs537601629, rs537604152, rs537613333, rs537617778, rs537631785,rs537633181, rs537633935, rs537639618, rs537667034, rs537667609,rs537669803, rs537670157, rs537670667, rs537672333, rs537682364,rs537682369, rs537686133, rs537687224, rs537687568, rs537695131,rs537703078, rs537707108, rs537710357, rs537710408, rs537712643,rs537715467, rs537718154, rs537723004, rs537737178, rs537746735,rs537754618, rs537761624, rs537764120, rs537767174, rs537776013,rs537777122, rs537780080, rs537780353, rs537793909, rs539458219,rs539458518, rs539465004, rs539483957, rs539487721, rs539490176,rs539490713, rs539493737, rs539509844, rs539513572, rs539515043,rs539517115, rs539520506, rs539520921, rs539547411, rs539555236,rs539557415, rs539560146, rs539561125, rs539562912, rs539568123,rs539575854, rs539588816, rs539590212, rs539594583, rs539600108,rs539613256, rs539637667, rs539639483, rs539646508, rs539647958,rs539649405, rs539651003, rs539654480, rs539659107, rs539667537,rs539668482, rs539681057, rs539685891, rs539691275, rs539703515,rs539709834, rs539727134, rs539728586, rs539737972, rs539743044,rs539744402, rs539746889, rs539747838, rs539749078, rs539752287,rs539753975, rs540056477, rs540058092, rs540060103, rs540064241,rs540068366, rs540075059, rs540076314, rs540082133, rs540085209,rs540088670, rs540097341, rs540101289, rs540103484, rs540113508,rs540120801, rs540123837, rs540124979, rs540138357, rs540139422,rs540140573, rs540144669, rs540166739, rs540167430, rs540170162,rs540176970, rs540210228, rs540210454, rs540217687, rs540223266,rs540229105, rs540238743, rs540244181, rs540245381, rs540250681,rs540253614, rs540263609, rs540269469, rs540273767, rs540279593,rs540296376, rs540319951, rs540335775, rs540337109, rs540340958,rs540343758, rs540346484, rs540348495, rs540363938, rs540366388,rs540366502, rs540367904, rs540381999, rs540390139, rs540390470,rs540393893, rs540406875, rs540407393, rs540409535, rs540419764,rs540424415, rs540434717, rs540435445, rs540441760, rs540449411,rs540459659, rs540471823, rs540472265, rs540478764, rs540478766,rs540487524, rs540491207, rs540499042, rs540509185, rs540521147,rs540524310, rs540530272, rs540530546, rs540531472, rs540540417,rs540552029, rs540555244, rs540558324, rs540570959, rs540580875,rs540583356, rs540601562, rs540603896, rs540606586, rs540615102,rs540617635, rs540620598, rs540647551, rs540649294, rs540650670,rs540654176, rs540654480, rs540657498, rs540668129, rs540675789,rs540676786, rs540688007, 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rs541107031,rs541109722, rs541116304, rs541123286, rs541131051, rs541242485,rs541246463, rs541246634, rs541252917, rs541264425, rs541270121,rs541271797, rs541290371, rs541292740, rs541296277, rs541298920,rs541320020, rs541329767, rs541337098, rs541340603, rs541346666,rs541361311, rs541361798, rs541363701, rs541366793, rs541403261,rs541404870, rs541414966, rs541416694, rs541418868, rs541424781,rs541432385, rs541437069, rs541446630, rs541448855, rs541449946,rs541461413, rs541464425, rs541464496, rs541469509, rs541484448,rs541489057, rs541489362, rs541501043, rs541506154, rs541510486,rs541515957, rs541522635, rs541523715, rs541524474, rs541531137,rs541539536, rs541563581, rs541575271, rs541581476, rs541590032,rs541593610, rs541595385, rs541596111, rs541600509, rs541609677,rs541620387, rs541631722, rs541633118, rs541654487, rs541658128,rs541664161, rs541665175, rs541686643, rs541688747, rs541690936,rs541691442, rs541694511, rs541714486, rs541715202, rs541720549,rs541722240, rs541723658, rs541724809, rs541729928, rs541730001,rs541738401, rs541747926, rs541756329, rs541757928, rs541759486,rs541759694, rs541766895, rs541767288, rs541783476, rs541791267,rs541792064, rs541794184, rs541810036, rs541819630, rs541820776,rs541829831, rs541837687, rs541839149, rs541848475, rs541851615,rs541858851, rs541860427, rs541863192, rs541866153, rs541866364,rs541867611, rs541876402, rs541876449, rs541877062, rs541877660,rs541878085, rs541883153, rs541883710, rs541883838, rs541886502,rs541889873, rs541906693, rs541909799, rs543992564, rs541913217,rs541914850, rs541922944, rs541932773, rs541936544, rs541946478,rs541949499, rs541972808, rs541974579, rs541975342, rs541996661,rs541999721, rs541999748, rs542009150, rs542012284, rs542012370,rs542017100, rs542017531, rs542018418, rs542031004, rs542039403,rs542040543, rs542041929, rs542048480, rs542048549, rs542069112,rs542077369, rs542083792, rs542098436, rs542111979, rs542123047,rs542143143, rs542143662, rs542153522, rs542158128, rs542163807,rs542167553, rs542177479, rs542181026, rs542181156, rs542186753,rs542189754, rs542190269, rs539754160, rs539755401, rs539760951,rs539773828, rs539774645, rs539792219, rs539796955, rs539797469,rs539800334, rs539801818, rs539808786, rs539822437, rs539828624,rs539831755, rs539842606, rs539846544, rs539853017, rs539863558,rs539871944, rs539878231, rs539880192, rs539884273, rs539886538,rs539886792, rs539887350, rs539889958, rs539890398, rs539901235,rs539901904, rs539922983, rs539925157, rs539926601, rs539928574,rs539934981, rs539952086, rs539962761, rs539963965, rs539964979,rs539967734, rs539972064, rs539985566, rs540014841, rs540017634,rs540020784, rs540024877, rs540030879, rs540036475, rs540046467,rs540046692, rs540047580, rs542201666, rs542212244, rs542220390,rs542223373, rs542224295, rs542225769, rs542225985, rs542228814,rs542231131, rs542233313, rs542236715, rs542250211, rs542254852,rs542255519, rs542258416, rs542258527, rs542289170, rs542290881,rs542298518, rs542316464, 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rs542964486,rs542969480, rs542989319, rs542991953, rs543003828, rs543007476,rs543017965, rs543028914, rs543028950, rs543033687, rs543037517,rs543038224, rs543038274, rs543052310, rs543060376, rs543063739,rs543064556, rs543070097, rs543076202, rs543081201, rs543087175,rs543091166, rs543097478, rs543100622, rs543107459, rs543110305,rs543118251, rs543127958, rs543133516, rs543142662, rs543143075,rs543149667, rs543151567, rs543153056, rs543171256, rs543171979,rs543175610, rs543192890, rs543193964, rs543198342, rs543200239,rs543201170, rs543219034, rs543227967, rs543236498, rs543239168,rs543242168, rs543245009, rs543245860, rs543249800, rs543253432,rs543261959, rs543268915, rs543274695, rs543283947, rs543296901,rs543299238, rs543299804, rs543319666, rs543325134, rs543338815,rs541133099, rs541136875, rs541138598, rs541153108, rs541160920,rs541187208, rs541192886, rs541218031, rs541232828, rs541237035,rs541241195, rs541241809, rs543349542, rs543351855, rs543353830,rs543368331, rs543369774, rs543374158, rs543374763, rs543379501,rs543396580, rs543402621, rs543405955, rs545666176, rs543407275,rs543411735, rs543415094, rs543418643, rs543422357, rs543431544,rs543450716, rs543456495, rs543456737, rs543459342, rs543461652,rs543473539, rs543473852, rs543478361, rs543481138, rs543491384,rs543499084, rs543505575, rs543517372, rs543519833, rs543539626,rs543542609, rs543542771, rs543543141, rs543556031, rs543565820,rs543575441, rs543576848, rs543581388, rs543585282, rs543605066,rs543606449, rs543612531, rs543612739, rs543631144, rs543634241,rs543635294, rs543638415, rs543639665, rs543640204, rs543648267,rs543650014, rs543650977, rs543661072, rs543672452, rs543678283,rs543681221, rs543683035, rs543683708, rs543686769, rs543696015,rs543700068, rs543702737, rs543705106, rs543709216, rs543728046,rs543733837, rs543742166, rs543745212, rs543747761, rs543751867,rs543754533, rs543757260, rs543774199, rs543774491, rs543783982,rs543793030, rs543793520, rs543798898, rs543799983, 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rs544277816, rs544284738, rs544289374,rs544294119, rs544299574, rs544302004, rs544304573, rs544304599,rs544307319, rs544328684, rs544329981, rs544334861, rs544343139,rs544348942, rs544349148, rs544349338, rs544357697, rs544360921,rs544366864, rs544376534, rs544382960, rs544391550, rs544424901,rs544427039, rs544428305, rs544428390, rs544431914, rs544432455,rs544436206, rs544437502, rs544449605, rs544450143, rs544457039,rs544457791, rs544460707, rs544476414, rs544479054, rs544480269,rs544486386, rs544486433, rs544491067, rs544503029, rs544512597,rs544516739, rs544525358, rs544528477, rs544530054, rs544541633,rs544551844, rs544557865, rs544559788, rs544561022, rs544564998,rs544565430, rs546856906, rs546872203, rs544570513, rs544586814,rs544614771, rs544623038, rs544624137, rs544631967, rs544643689,rs544649034, rs544657091, rs544662168, rs544664410, rs544671667,rs544677075, rs544693480, rs544702226, rs546958863, rs546961693,rs546961936, rs546969706, rs544707773, rs544717742, rs544721571,rs544725284, rs544725616, rs544726392, rs544735499, rs544737326,rs544742641, rs544742705, rs544743996, rs544745290, rs544745745,rs544751630, rs544756792, rs544757437, rs544761167, rs544762113,rs544780902, rs544792156, rs544802918, rs544810475, rs544813750,rs544813807, rs544835582, rs544853905, rs542495368, rs542503058,rs542507652, rs542517359, rs542522903, rs542524211, rs542525674,rs542528931, rs542534795, rs542540054, rs542547224, rs542550048,rs542553336, rs542569515, rs542573469, rs542578446, rs542578646,rs542601520, rs542603251, rs542610673, rs544854140, rs544856827,rs544865440, rs544880920, rs544882270, rs544882565, rs544887019,rs544897041, rs544897324, rs544902509, rs544911518, rs544913633,rs544914643, rs544914728, rs544916955, rs544920067, rs544920343,rs544926919, rs544932690, rs544946192, rs544948202, rs544949193,rs544950788, rs544955377, rs544961646, rs544962466, rs544971227,rs544975075, rs544992392, rs544999397, rs545000422, rs545006403,rs545009138, rs545010053, 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rs552724124, rs552725949, rs552729404,rs552732177, rs552750166, rs552751755, rs552753274, rs552755081,rs552764919, rs552769057, rs552777664, rs552777938, rs552809287,rs552833340, rs552841304, rs552844142, rs552846531, rs552848730,rs552849999, rs552855782, rs552859272, rs552861705, rs552862912,rs552865010, rs552881111, rs552889582, rs552907117, rs552922080,rs552932201, rs552934726, rs552939049, rs552943170, rs552945812,rs552959442, rs552960571, rs552961366, rs552970366, rs552973039,rs552973880, rs552978096, rs553000644, rs553001789, rs553020425,rs553024743, rs553033904, rs553037566, rs553052815, rs553056705,rs553059764, rs553066491, rs553080917, rs553082174, rs553082225,rs553091459, rs553100950, rs553101419, rs553103591, rs553111479,rs553119101, rs553124392, rs553125135, rs553125571, rs553132462,rs553134755, rs553137805, rs553144168, rs553151115, rs553154019,rs553183444, rs553188831, rs553196736, rs553205277, rs553213219,rs553213953, rs553227187, rs553228517, rs553228780, rs553240147,rs553245207, rs553246435, rs553249463, rs553251150, rs553251873,rs553254729, rs553264130, rs553265266, rs553268970, rs553271535,rs553274109, rs553274137, rs553278237, rs553280785, rs553285981,rs553290339, rs553309499, rs553327821, rs553345309, rs553348430,rs553349512, rs553353724, rs553357782, rs553374507, rs553376568,rs553382039, rs553384697, rs553387704, rs553390510, rs553397587,rs553411669, rs553412432, rs553419493, rs553449827, rs553469921,rs553480256, rs553502202, rs553511552, rs553512693, rs553534938,rs553537396, rs553546148, rs553548534, rs553565332, rs553568005,rs553577161, rs553581722, rs553582559, rs553587528, rs553590642,rs553591315, rs553596309, rs553599911, rs553605064, rs553620454,rs553623284, rs553633355, rs553637587, rs553646799, rs553652958,rs553653736, rs553657770, rs553665046, rs553668059, rs553731959,rs555937825, rs553736788, rs553741097, rs553741186, rs553751174,rs553752296, rs553754496, rs553760687, rs553762213, rs553767654,rs553771239, rs553777899, rs553786524, rs553812511, rs553813130,rs553816154, rs553820800, rs553821057, rs553830643, rs553834272,rs553840298, rs553859164, rs553868520, rs553870362, rs553875418,rs553875987, rs553894903, rs553896504, rs553897835, rs553901105,rs553902324, rs553902689, rs553907868, rs553909391, rs553911386,rs553914862, rs553923445, rs553937637, rs553942807, rs553942963,rs553950374, rs553955264, rs553957310, rs553964242, rs553964307,rs553967707, rs553974541, rs553975674, rs553981077, rs553985618,rs554008041, rs554011769, rs554015484, rs554025892, rs554029938,rs554034391, rs554036355, rs554053486, rs554053828, rs554055140,rs554057836, rs554058474, rs554068660, rs554092022, rs554100752,rs554101095, rs554107427, rs554124528, rs554127338, rs554127885,rs554132123, rs554133004, rs551757963, rs551762824, rs551763302,rs551764034, rs551776824, rs551776952, rs551780407, rs551788004,rs551793068, rs551793997, rs551796722, rs551800034, rs551808150,rs551813444, rs551815642, rs551817171, rs551833923, rs551835408,rs551838790, rs551840838, rs551843401, rs551846101, rs551849407,rs551858292, rs551861310, rs556578702, rs551864368, rs551873276,rs551881679, rs551882069, rs551887296, rs551892793, rs554136284,rs554136358, rs554138287, rs554140205, rs554156773, rs554157713,rs554158251, rs554169280, rs554170002, rs554171667, rs554175309,rs554176057, rs554179538, rs554192723, rs554192934, rs554202420,rs554235539, rs554239896, rs554254676, rs554262972, rs554274005,rs554291900, rs554300952, rs554301748, rs554303224, rs554303424,rs554304739, rs554306624, rs554319942, rs554323764, rs554326408,rs554326656, rs554343700, rs554356517, rs554389229, rs554389746,rs554392438, rs554392640, rs554393112, rs554396631, rs554401158,rs554407038, rs554412253, rs554434688, rs554436861, rs554439409,rs554439522, rs554442898, rs554449195, rs554453904, rs554458333,rs554463011, rs554469478, rs554473207, rs554480203, rs554490230,rs554495126, rs554507156, rs554511544, rs554525541, rs554527474,rs554529892, rs554542294, rs554542885, rs554548547, rs554555563,rs554564434, rs554566607, rs554581974, rs554592085, rs554595627,rs554603112, rs554608720, rs554629878, rs554631498, rs554639059,rs554643242, rs554644153, rs554647298, rs554653439, rs554662567,rs554664825, rs554675358, rs554675732, rs554683446, rs554695978,rs554696709, rs554699678, rs554699818, rs554703480, rs554706659,rs554724524, rs554728117, rs554731796, rs554731971, rs554836852,rs554839192, rs554861167, rs554866928, rs554874271, rs554875497,rs554875624, rs554890518, rs554892246, rs554906204, rs554919116,rs554924203, rs554925354, rs554929541, rs554935444, rs554941274,rs554950949, rs554954077, rs554957578, rs554958483, rs554961431,rs554961535, rs554963755, rs554966876, rs554981595, rs555002241,rs555004494, rs555005660, rs555015357, rs555023999, rs555031713,rs555033634, rs555192114, rs555192184, rs555204659, rs555213485,rs555218136, rs555218357, rs555219395, rs555224930, rs555228742,rs555228979, rs555233578, rs555243038, rs555243372, rs555252612,rs555257049, rs555275223, rs555279276, rs555281777, rs555498033,rs555498175, rs555499218, rs555515930, rs555531598, rs555547011,rs555547387, rs555554254, rs555556426, rs555556597, rs555578445,rs555585631, rs555595260, rs555598372, rs555610613, rs555617744,rs555630717, rs555637984, rs555638163, rs555640676, rs555654312,rs555662381, rs555674246, rs555675329, rs555683808, rs555685049,rs555692568, rs555692596, rs555716212, rs555722086, rs555723716,rs555729345, rs555735447, rs553671738, rs553673913, rs553690724,rs553691701, rs553694442, rs553694824, rs553705075, rs553721801,rs553723206, rs553728368, rs555941765, rs555945326, rs555962103,rs555974343, rs555974910, rs555977921, rs555980865, rs555981051,rs555983669, rs555986447, rs555988253, rs556022415, rs556025562,rs556026825, rs556029311, rs556033610, rs556042808, rs556053135,rs556059021, rs556069082, rs556069654, rs556082532, rs556093467,rs556100797, rs556103068, rs556103716, rs556123793, rs556143290,rs556144819, rs556147181, rs556149044, rs556151817, rs556158433,rs556158703, rs556178449, rs556178706, rs556181500, rs556190674,rs556196230, rs556197182, rs556206258, rs556217346, rs556255149,rs556264016, rs556264495, rs556279897, rs556290497, rs556295173,rs556296338, rs556299266, rs556312482, rs556317104, rs556321738,rs556326290, rs556331086, rs556331550, rs556332896, rs556335217,rs556335656, rs556338839, rs556345825, rs556354956, rs556356576,rs556363117, rs556367685, rs556369578, rs556376813, rs556379100,rs556388155, rs556392808, rs556397294, rs556399318, rs556400622,rs556412700, rs556416030, rs556422723, rs556424092, rs556433094,rs556438775, rs556452892, rs556498565, rs556509282, rs556512845,rs556515522, rs556516868, rs556525848, rs556534004, rs556536576,rs556536690, rs556538506, rs556552701, rs556553141, rs556561100,rs556562607, rs556568140, rs556570597, rs556580040, rs556604716,rs556607793, rs556631925, rs556654590, rs556674208, rs556678279,rs556681576, rs556686601, rs556693645, rs556704400, rs556705268,rs556713600, rs556716735, rs556732394, rs556734401, rs556742141,rs556744288, rs556761236, rs556780033, rs556792121, rs556794307,rs556796191, rs556799646, rs556805739, rs556806428, rs556808385,rs556813110, rs556831347, rs556832492, rs556835813, rs556835940,rs556850166, rs556858845, rs556867060, rs556873801, rs556875669,rs556877578, rs556885026, rs556905043, rs556906553, rs556907329,rs556910489, rs556913714, rs556917622, rs558843830, rs556923906,rs556926153, rs556926852, rs556943847, rs556952680, rs556955030,rs556962154, rs556970222, rs556975664, rs556983983, rs556985585,rs556988455, rs556992176, rs556995649, rs557008555, rs557010410,rs557010728, rs557012808, rs557018689, rs557019602, rs557024457,rs557027138, rs557036226, rs557046132, rs557046561, rs557068608,rs557071958, rs557075965, rs557076959, rs557078674, rs557084007,rs557091269, rs557092814, rs557097739, rs557102330, rs557130133,rs557134871, rs557135906, rs557143264, rs557163701, rs557176775,rs557177682, rs557179541, rs557180668, rs557180711, rs557182088,rs557183765, rs557189227, rs557197114, rs557206031, rs557210081,rs557213957, rs557217235, rs557222006, rs557222472, rs557227027,rs557229186, rs554749394, rs554751014, rs554774893, rs554779708,rs554779757, rs554785274, rs554802570, rs554804124, rs554806091,rs554817715, rs554826900, rs554831706, rs554836323, rs557245021,rs557245119, rs557252193, rs557252909, rs557258333, rs557270342,rs557275941, rs557291752, rs557294288, rs557307162, rs557307768,rs557312139, rs557334458, rs557341253, rs557341379, rs557368113,rs557388817, rs557389288, rs557390439, rs557396252, rs557396395,rs557414016, rs557414967, rs557417588, rs557438685, rs557442137,rs557454149, rs557457027, rs557464999, rs557470989, rs557475403,rs557484326, rs555035616, rs555039593, rs555048761, rs555049181,rs555049397, rs555050802, rs555060023, rs555062062, rs555065139,rs555066436, rs555070617, rs555085484, rs555085711, rs555089084,rs555091296, rs555105213, rs555123382, rs555124061, rs555125567,rs555143486, rs555144351, rs555146927, rs555155503, rs555156628,rs555160600, rs555178901, rs555179247, rs555191897, rs557541319,rs557543919, rs557548994, rs557550799, rs557553913, rs557556658,rs557571344, rs557577794, rs557590945, rs557608400, rs557609768,rs557614270, rs557621475, rs557626363, rs557631916, rs557636917,rs557643974, rs557650512, rs555295201, rs555319319, rs555322259,rs555323474, rs555325135, rs555330633, rs555333669, rs555336285,rs555338197, rs555341419, rs555344901, rs555358449, rs555359581,rs555362325, rs555365950, rs555367409, rs555370725, rs555374988,rs555394767, rs555394805, rs555395818, rs555419439, rs555421401,rs555428489, rs555428583, rs555430144, rs555431578, rs555438522,rs555449284, rs555449945, rs555455023, rs555457218, rs555460812,rs555468485, rs555472242, rs555475215, rs555492804, rs557656432,rs557658018, rs557661141, rs557666902, rs557669520, rs557672569,rs557678799, rs557690968, rs557698053, rs557700762, rs557701211,rs557740831, rs557782178, rs557791691, rs557792602, rs557797781,rs557807103, rs557809394, rs557815112, rs557819748, rs557825492,rs557829722, rs557856382, rs557859788, rs557864469, rs557866480,rs557866975, rs555736263, rs555740540, rs555743930, rs555750520,rs555750527, rs555768755, rs555768863, rs555780473, rs555789684,rs555793502, rs555797671, rs555800243, rs555813804, rs555823247,rs555825927, rs555826030, rs555833747, rs555863641, rs555865358,rs555877088, rs555880987, rs555891640, rs555894096, rs555896938,rs555901841, rs555902751, rs555912003, rs555913429, rs555930271,rs555930529, rs555934080, rs557868669, rs557870922, rs557878734,rs557879521, rs557886284, rs557886285, rs557890903, rs557918048,rs557918369, rs557927915, rs557939977, rs557945426, rs557951683,rs557953170, rs557959784, rs557963905, rs557964964, rs557965133,rs557965588, rs557966248, rs557968171, rs557988734, rs557991078,rs557993433, rs558009786, rs558010892, rs558012608, rs558027602,rs558050775, rs558057320, rs558061387, rs558067980, rs558076110,rs558076966, rs558077622, rs558086266, rs558088963, rs558098380,rs558107130, rs558111125, rs558115670, rs558117607, rs558121023,rs558137007, rs558141828, rs558145183, rs558165786, rs558169766,rs558175255, rs558181380, rs558208331, rs558214162, rs558224430,rs558224513, rs558225482, rs558233277, rs558245703, rs558247119,rs558251182, rs558254661, rs558260693, rs558274851, rs558277318,rs558283476, rs558291247, rs558296625, rs558309234, rs558316870,rs558334476, rs558334622, rs558347296, rs558348138, rs558353508,rs558360153, rs558372267, rs558377779, rs558386929, rs558389080,rs558391623, rs558409197, rs558409397, rs558409777, rs558421053,rs558422246, rs558423088, rs558427297, rs558428767, rs558429730,rs558432480, rs558438888, rs558441799, rs558445250, rs558445370,rs558450460, rs558453249, rs558454887, rs558471349, rs558476731,rs558480408, rs558480436, rs558484138, rs558496621, rs558503815,rs558505450, rs558514541, rs558515354, rs558522635, rs558524886,rs558545224, rs558548885, rs558562386, rs558566808, rs558585973,rs558600464, rs558607782, rs558617675, rs558627630, rs558629668,rs558635401, rs558657635, rs558658211, rs558664687, rs558673965,rs558676511, rs558685087, rs558693403, rs558704045, rs558706170,rs558716821, rs558718569, rs558729382, rs558739358, rs558743902,rs558750809, rs558758069, rs558761155, rs558764736, rs558766107,rs558772103, rs558787436, rs558795536, rs558806244, rs558808487,rs558816828, rs558829754, rs558839023, rs558843539, rs558844479,rs558848311, rs558848903, rs558850283, rs558851376, rs558851493,rs558855359, rs558855698, rs558857156, rs558862164, rs558866950,rs558873373, rs558884037, rs558893405, rs558893829, rs558893900,rs558894632, rs558895294, rs558920876, rs558922407, rs558946959,rs558977022, rs558985150, rs558988770, rs559010844, rs559015689,rs559016765, rs559025347, rs559026649, rs559030911, rs559034857,rs559037190, rs559042927, rs559049164, rs559050452, rs559051135,rs559055995, rs559061812, rs559066591, rs559067472, rs559081511,rs559086442, rs559087862, rs559090131, rs559091055, rs559100697,rs559105328, rs559108754, rs559125068, rs559127996, rs559136492,rs559138713, rs559142936, rs559143265, rs559143927, rs559149262,rs559156639, rs559160266, rs559171614, rs559177744, rs559180276,rs559180551, rs559198733, rs559199621, rs559203592, rs559208688,rs559212594, rs559213895, rs559217118, rs559218497, rs559221339,rs559230542, rs559238341, rs559239968, rs559241886, rs559259561,rs561498621, rs561516953, rs559261471, rs559268835, rs559286506,rs559293005, rs559293390, rs559293651, rs559300745, rs559301408,rs559306817, rs559314593, rs559316602, rs559321052, rs559322596,rs559330429, rs559330521, rs559332291, rs559339075, rs559339724,rs559354130, rs559357973, rs559360564, rs559373517, rs559375956,rs559384876, rs559388947, rs559393729, rs559395057, rs559397483,rs559409371, rs559409587, rs559411567, rs559416445, rs559427490,rs559428648, rs559441070, rs559441152, rs559462297, rs559462613,rs559462832, rs559467158, rs559471816, rs559479414, rs559511783,rs559513908, rs559514972, rs559518867, rs559519784, rs559521734,rs559532856, rs559545211, rs559552432, rs559553893, rs559561414,rs557710906, rs557711504, rs557720168, rs557726549, rs557728819,rs557729697, rs557903160, rs557903411, rs557915713, rs560317867,rs560323261, rs560326117, rs560330487, rs560338206, rs560343203,rs560360895, rs560362734, rs560371375, rs561949216, rs560386774,rs560390937, rs560396355, rs560397833, rs560398408, rs560398621,rs560402607, rs560404582, rs560411245, rs560418276, rs560424660,rs560428220, rs560428299, rs560438080, rs560442647, rs560447310,rs560447432, rs560456728, rs560456765, rs560456801, rs560463347,rs560463738, rs560470786, rs560476842, rs560479945, rs560505619,rs560516167, rs560519220, rs560541292, rs560552544, rs560552575,rs560562456, rs560580888, rs560581866, rs560585152, rs560590672,rs560592598, rs560607321, rs560618482, rs560626777, rs560629883,rs560639434, rs560642352, rs560643364, rs560644296, rs560650031,rs560652517, rs560661386, rs560661728, rs560671913, rs560679732,rs560687509, rs560687841, rs560692645, rs560694995, rs560697920,rs560705108, rs560711713, rs560716762, rs560728311, rs560729719,rs560730023, rs560735908, rs560738783, rs560742895, rs560750730,rs560753415, rs560756966, rs560761359, rs560768338, rs560769232,rs560772341, rs560775060, rs560953239, rs560958307, rs560970569,rs560973110, rs560974790, rs560977669, rs560985102, rs560988217,rs560996286, rs560997570, rs560998592, rs561003121, rs561005604,rs561013078, rs561014885, rs561031853, rs561037892, rs561039511,rs561050122, rs561051609, rs561054890, rs561069219, rs561070695,rs561080010, rs561086278, rs561087452, rs561088363, rs561094343,rs561099203, rs561103070, rs561107911, rs561108229, rs561116559,rs561119570, rs561122879, rs561137872, rs561141146, rs561141685,rs561154698, rs561160774, rs561162163, rs561174849, rs561178157,rs561192905, rs561207461, rs561207863, rs561208033, rs561216181,rs561221720, rs561228831, rs561230427, rs561231996, rs561245603,rs561247557, rs561250324, rs561270068, rs561275191, rs561278586,rs561283086, rs561283405, rs561283598, rs561297150, rs561304717,rs561308515, rs561309127, rs561315148, rs561318582, rs561320700,rs561343463, rs561351555, rs561352213, rs561352499, rs561354505,rs561354947, rs561356937, rs561359423, rs561360752, rs561363001,rs561364929, rs561383425, rs561385467, rs561388735, rs561391210,rs561395044, rs561395786, rs561402432, rs561402579, rs561406742,rs561415312, rs561416234, rs561418451, rs561422068, rs561427935,rs561432263, rs561439256, rs561442495, rs561461451, rs561462132,rs561474991, rs561478062, rs561478810, rs561479359, rs561483164,rs561498581, rs561520081, rs561525437, rs561527252, rs561539443,rs561551192, rs561552667, rs561561151, rs561563685, rs561564761,rs561565268, rs561583528, rs561583603, rs561602561, rs561626708,rs561631652, rs561640190, rs561640362, rs561651683, rs561652781,rs561663374, rs561663686, rs561671523, rs561671731, rs561671751,rs561680234, rs561690791, rs561696442, rs561706782, rs561707257,rs561710553, rs559565545, rs559570937, rs559574430, rs559584857,rs559587986, rs559592944, rs559601674, rs559607703, rs559608999,rs559609408, rs559611858, rs559633989, rs559644796, rs559646667,rs559654152, rs559660587, rs559661776, rs559667794, rs559668827,rs559669453, rs559671684, rs559695052, rs559695606, rs559696780,rs559700486, rs559731481, rs559739755, rs559758075, rs559762627,rs559762724, rs559766224, rs559767768, rs559775286, rs559778243,rs559786431, rs559788065, rs559788169, rs559789847, rs559794730,rs559795013, rs559798155, rs559798757, rs559799668, rs559801429,rs559802100, rs559827192, rs559828383, rs559840482, rs559853310,rs559855924, rs559860398, rs559860618, rs559874582, rs559881805,rs559889941, rs559897146, rs559902232, rs559910998, rs559914606,rs559918764, rs559921882, rs559924343, rs559931396, rs559933288,rs559935097, rs559946335, rs559947559, rs559950813, rs559958932,rs559970433, rs559982345, rs559984034, rs559985796, rs559989442,rs559995131, rs559996104, rs560003272, rs560007200, rs560009774,rs560016803, rs560025077, rs560031891, rs560037633, rs560042504,rs560069645, rs560078993, rs560080329, rs560097440, rs560098353,rs560100548, rs560125745, rs560132430, rs560133398, rs560138292,rs560149323, rs560161885, rs560163591, rs560168634, rs560170353,rs560171915, rs560173696, rs560185622, rs560194741, rs560195969,rs560200894, rs560202831, rs560205662, rs560205891, rs560236302,rs560242507, rs560250817, rs560260655, rs560265434, rs560267692,rs560268068, rs560275717, rs560279265, rs560288161, rs560300791,rs560303142, rs560304265, rs560312665, rs561913290, rs561918333,rs561922909, rs561923042, rs561926004, rs561926363, rs561927660,rs561938850, rs561957357, rs561967083, rs561971273, rs561972788,rs561983984, rs561995503, rs562003075, rs562003174, rs562003307,rs562004911, rs562009949, rs562012670, rs562020637, rs562036355,rs562060490, rs562060959, rs562064148, rs562066279, rs562077385,rs562079102, rs562079361, rs562089379, rs562089614, rs562106833,rs562112000, rs562114543, rs562121376, rs562128363, rs562130977,rs562132282, rs562133851, rs562136005, rs562136121, rs562136528,rs562147875, rs562152962, rs562172831, rs562175467, rs562176626,rs562190702, rs562196785, rs562197009, rs562197054, rs562198118,rs562201810, rs562210666, rs562216982, rs562220572, rs562234846,rs562249604, rs562250614, rs562251535, rs562252124, rs562253530,rs562273422, rs562279095, rs562280765, rs562291251, rs562298902,rs562310277, rs562310493, rs562320544, rs562320968, rs562323206,rs562325735, rs562329242, rs562331184, rs562334852, rs562340968,rs562341234, rs562346872, rs562355238, rs562359226, rs562363257,rs560793996, rs560801433, rs560813379, rs560819927, rs560820038,rs560820630, rs560821251, rs560824508, rs560824710, rs560830305,rs560834832, rs560843715, rs560847295, rs560849930, rs560862196,rs560867368, rs560875573, rs560875747, rs560876227, rs560877337,rs560882531, rs560884968, rs560885345, rs560912177, rs560929019,rs560934411, rs560940006, rs560940942, rs560952700, rs562363344,rs562364279, rs562369962, rs562380847, rs562384370, rs562399529,rs562403069, rs562414473, rs562416693, rs562442240, rs562451183,rs562451345, rs562453989, rs562456146, rs562462720, rs562471509,rs562475359, rs562487445, rs562487782, rs562491752, rs562493912,rs562501188, rs562502230, rs562510888, rs562510918, rs562519109,rs564771228, rs562532483, rs562533987, rs562537713, rs562551927,rs562562895, rs562562993, rs562563001, rs562573217, rs562573852,rs562581878, rs562595941, rs562596557, rs562597002, rs562604859,rs562605128, rs562607108, rs562617792, rs562627959, rs562628736,rs562631480, rs562633099, rs562637683, rs562640599, rs562642520,rs562645168, rs562663555, rs562672120, rs562677251, rs562688878,rs562694736, rs562697529, rs562715666, rs562725955, rs562727481,rs562728546, rs562746520, rs562755889, rs562757567, rs562759979,rs562760691, rs562762668, rs562763128, rs562769214, rs562802059,rs562807680, rs562811901, rs562820032, rs562821251, rs562828276,rs562829872, rs562833114, rs562834227, rs562835391, rs562836201,rs562851594, rs562854591, rs562864964, rs562881136, rs562882121,rs562886974, rs562892571, rs562894571, rs562913482, rs562915926,rs562916592, rs562918247, rs562940221, rs562940441, rs562947172,rs562954973, rs562964677, rs562967724, rs562983310, rs562986722,rs562992103, rs562993175, rs563001052, rs563008409, rs563010068,rs563024126, rs563026226, rs563047937, rs563062090, rs563068353,rs563068817, rs563075043, rs563080784, rs563084805, rs563091092,rs563107076, rs563112152, rs563114760, rs563117678, rs563154120,rs563154293, rs563156340, rs563188057, rs563192813, rs563202027,rs563204320, rs563209804, rs563221062, rs563222937, rs563227989,rs563231745, rs563232703, rs563253783, rs563258058, rs561713039,rs561715264, rs561716438, rs561717679, rs561720959, rs561721003,rs561723845, rs561740296, rs561743780, rs561752997, rs561755732,rs561764385, rs561778100, rs561779334, rs561781260, rs561795776,rs561800476, rs561800700, rs561801248, rs561821767, rs561831123,rs561832533, rs561839838, rs561841716, rs563260191, rs563261199,rs563262466, rs563263421, rs563305414, rs565587725, rs565591911,rs565596444, rs563306697, rs563309398, rs563314225, rs563315555,rs563316556, rs563318639, rs563326184, rs563346430, rs563349989,rs563352798, rs563352821, rs563375190, rs563377220, rs563387613,rs563392001, rs563402502, rs563424578, rs563425665, rs563426554,rs563426697, rs563427455, rs563427718, rs563436453, rs563451668,rs563455173, rs563460690, rs563476394, rs563479057, rs563486128,rs563505578, rs563506483, rs563528050, rs563529543, rs563531546,rs563532261, rs563538726, rs563543177, rs563546527, rs563561487,rs563566452, rs563594168, rs563594639, rs563600079, rs563600962,rs563602225, rs563613654, rs563616896, rs563637424, rs563644478,rs563650562, rs561851943, rs561857532, rs561859867, rs561869286,rs561875939, rs561878232, rs561885794, rs561886071, rs561903702,rs564007420, rs564017003, rs564017979, rs564019104, rs564021907,rs564029136, rs564033201, rs564037559, rs564045752, rs564051675,rs564056787, rs564065081, rs564076255, rs564082835, rs564093964,rs564095518, rs564100327, rs564106701, rs564107942, rs564112260,rs564113353, rs564122230, rs564127597, rs564128116, rs564140518,rs564144866, rs564148257, rs564150096, rs564161004, rs564163568,rs564163967, rs564168910, rs564171526, rs564172524, rs564181352,rs564188431, rs564198162, rs564204956, rs564209634, rs564237924,rs564241138, rs564248244, rs564260796, rs564262939, rs564265723,rs564278418, rs564280986, rs564282003, rs564290281, rs564307534,rs564308828, rs564312015, rs564316792, rs564329569, rs564340391,rs564340757, rs564340939, rs564346072, rs564350388, rs564358263,rs564358476, rs564376664, rs564380761, rs564381484, rs564385687,rs564395083, rs564396192, rs564398468, rs564404936, rs564411730,rs564416310, rs564418832, rs564419076, rs564426261, rs564435727,rs564449973, rs564453869, rs564458890, rs564459808, rs564461491,rs564468820, rs564469527, rs564473249, rs564473703, rs564474501,rs564474702, rs564475257, rs564478118, rs564493100, rs564505504,rs564508590, rs564509901, rs564536899, rs564542578, rs564542948,rs564556654, rs564573853, rs564573947, rs564597083, rs564600829,rs564601382, rs564604981, rs564622001, rs564622154, rs564625543,rs564626948, rs564632096, rs564633968, rs564634765, rs564636250,rs564636302, rs564640354, rs564640432, rs564657473, rs564663955,rs564671373, rs564671639, rs564679668, rs564680095, rs564681418,rs564684354, rs564687938, rs564701914, rs564712422, rs564712537,rs564712765, rs564715924, rs564716940, rs564729495, rs564729870,rs564735408, rs564735958, rs564736557, rs564736880, rs564741191,rs564756874, rs564773630, rs564775962, rs564777085, rs564781495,rs564795788, rs564813153, rs564817450, rs564827041, rs564834939,rs564842608, rs564857356, rs564858031, rs564858797, rs564866078,rs564867904, rs564874943, rs564889367, rs564892504, rs564893478,rs564896406, rs564898103, rs564900254, rs564905039, rs564917129,rs564922152, rs564928929, rs564935505, rs564949843, rs564950442,rs564955120, rs564967063, rs564968379, rs564972380, rs564998177,rs564999327, rs565005207, rs565012267, rs565015178, rs565015845,rs565029428, rs565041934, rs565061967, rs565063652, rs565076348,rs565076354, rs565084781, rs565086844, rs565088908, rs565105717,rs565108179, rs565120122, rs565125351, rs565127287, rs565129360,rs565137880, rs565142303, rs565150709, rs565150718, rs565150875,rs565168853, rs565182123, rs565185944, rs565193959, rs565194132,rs565205027, rs565214016, rs565215275, rs565216442, rs565217875,rs565221123, rs565228722, rs565236508, rs565246889, rs565248071,rs565249967, rs565250512, rs565253248, rs565256452, rs565262701,rs565283745, rs565288878, rs565297949, rs565303385, rs565304838,rs565313488, rs565320971, rs565324134, rs565333955, rs565337661,rs565342205, rs565359131, rs565366078, rs565368605, rs565369903,rs565376543, rs565384555, rs565390579, rs565399917, rs565400662,rs565405041, rs565412772, rs565415519, rs565417080, rs565417588,rs567390981, rs567404520, rs567406249, rs567406598, rs567411781,rs567418688, rs565417725, rs565423134, rs565426772, rs565427462,rs565433233, rs565435780, rs565436104, rs565442518, rs565446760,rs565447095, rs565452241, rs565461839, rs565466717, rs565481138,rs565481679, rs565482768, rs565491872, rs565492947, rs565503062,rs565505210, rs565514806, rs565520717, rs565523134, rs565537966,rs565538512, rs565553857, rs565555691, rs565557239, rs565557830,rs565565568, rs565578084, rs565582101, rs565584456, rs565600698,rs565601897, rs565615218, rs565616607, rs565617103, rs565622531,rs565622898, rs565628484, rs565633721, rs565634026, rs565642933,rs565645843, rs565660177, rs565664564, rs565667475, rs565676550,rs565695819, rs565696128, rs565706603, rs565720994, rs565733462,rs565735593, rs565747557, rs565750289, rs565759807, rs565768876,rs565775582, rs565780423, rs565780838, rs565782448, rs565786058,rs565787400, rs565787765, rs565798572, rs565801030, rs565811646,rs565811717, rs565813325, rs565819298, rs565832860, rs565836099,rs565840675, rs565841613, rs565844968, rs565845544, rs565845567,rs565846313, rs565848458, rs565879006, rs565884927, rs565885035,rs563651926, rs563654957, rs563655558, rs563658243, rs563660399,rs563663307, rs563673105, rs563680276, rs563685344, rs563689778,rs563690146, rs563690213, rs563696955, rs565887538, rs565887786,rs563709505, rs563711976, rs563728446, rs563733364, rs563737801,rs563746352, rs563748024, rs563773226, rs563773751, rs563779683,rs563790490, rs563791007, rs563800254, rs563807410, rs563810951,rs563815690, rs563818247, rs563818249, rs563820533, rs563835033,rs563838431, rs563838769, rs563838884, rs563844420, rs563852786,rs563852853, rs563853267, rs563859457, rs563861415, rs563863505,rs563873812, rs563887050, rs563889050, rs563891604, rs563895222,rs563896454, rs563907682, rs563914137, rs563916252, rs563922592,rs563930717, rs563940281, rs563940462, rs563942147, rs563944895,rs563953526, rs563960520, rs563966342, rs563969423, rs563974402,rs563974729, rs563991751, rs563997938, rs563998787, rs565937875,rs565950410, rs565955597, rs565961763, rs565965554, rs565968647,rs565972103, rs565976845, rs565978069, rs565992096, rs565994656,rs565998896, rs566000539, rs566017238, rs566022121, rs566022655,rs566024018, rs566028172, rs566030273, rs566037693, rs566050425,rs566054417, rs566059193, rs566061837, rs566066216, rs566066282,rs568536758, rs568537536, rs566074257, rs566078385, rs566078518,rs566088313, rs566089673, rs566094208, rs566102119, rs566106642,rs566112170, rs566132585, rs566139007, rs566146316, rs566154605,rs566156591, rs566162613, rs566165912, rs566169514, rs566174006,rs566182872, rs566185612, rs566194774, rs566202737, rs566207893,rs566209765, rs566211272, rs566212493, rs566220272, rs566228864,rs566230108, rs566233818, rs566243705, rs566244398, rs566261999,rs566263158, rs566268623, rs566268867, rs566269240, rs566273976,rs566274409, rs566284489, rs566289546, rs566300766, rs566318865,rs566321014, rs566323840, rs566324014, rs566328881, rs566330227,rs566333491, rs566351204, rs566355043, rs566356621, rs566371572,rs566374815, rs566379390, rs566394737, rs566411625, rs566417851,rs566430176, rs566434585, rs566437369, rs566449006, rs566454578,rs566456357, rs566462656, rs566470834, rs566485148, rs566486131,rs566486763, rs566487224, rs566491986, rs566497473, rs566502201,rs566518881, rs566535633, rs566542035, rs566549850, rs566555245,rs566588339, rs566603042, rs566605913, rs566610247, rs566611142,rs566614864, rs566623257, rs566624028, rs566633662, rs566635547,rs566636063, rs566636108, rs566644733, rs566647220, rs566661305,rs566661456, rs566662552, rs566670506, rs566675402, rs566680656,rs566697204, rs566699833, rs566701867, rs566702721, rs566702896,rs566707667, rs566708565, rs566722250, rs566727507, rs566732002,rs566744669, rs566745528, rs566749796, rs566753905, rs566767975,rs566775793, rs566781800, rs566786265, rs566787443, rs566794309,rs566794911, rs566797250, rs566802850, rs566804421, rs566824599,rs566829814, rs566840124, rs566843606, rs566850800, rs566850887,rs566854036, rs566857620, rs566867783, rs566875807, rs566876977,rs566882109, rs566893142, rs566899733, rs566908901, rs566914364,rs566916925, rs566918480, rs566919250, rs566919995, rs566932259,rs566942134, rs567177057, rs567177333, rs567189349, rs567194830,rs567198724, rs567201446, rs567206769, rs567212556, rs567220514,rs567239281, rs567243181, rs567247939, rs567264290, rs567266261,rs567272249, rs567279774, rs567284699, rs567285071, rs567297318,rs567302225, rs567305017, rs567317160, rs567327433, rs567327676,rs567352249, rs567369166, rs569547947, rs567376157, rs567376371,rs567387669, rs565906678, rs565920809, rs565928874, rs565930847,rs565932647, rs565935171, rs565936151, rs568409701, rs568417811,rs568428579, rs568433821, rs568443943, rs568448914, rs568459721,rs568460685, rs568461420, rs568465812, rs568467222, rs568467776,rs568480708, rs568495493, rs568496980, rs568497026, rs568497814,rs568505950, rs568506486, rs568510954, rs568518782, rs568523535,rs568526494, rs568532383, rs568535202, rs568544071, rs568544713,rs568576606, rs568581375, rs568581393, rs568581702, rs568585847,rs568587061, rs568587803, rs568600536, rs568609633, rs568615384,rs568619785, rs568625246, rs568631259, rs568636059, rs568638040,rs568643563, rs568643728, rs568647568, rs568649561, rs568650982,rs568655259, rs568658312, rs568662517, rs568677306, rs568678074,rs568681350, rs568688318, rs568690977, rs568702894, rs568717968,rs568724957, rs568725106, rs568735426, rs568741030, rs568742340,rs568756622, rs568758205, rs568762144, rs568767656, rs568770840,rs568771992, rs568773178, rs568774417, rs568797795, rs568806062,rs570028894, rs570029155, rs568827496, rs568828074, rs568833874,rs568837320, rs568838348, rs568841817, rs568844063, rs568860972,rs568880454, rs568883899, rs568884394, rs568887296, rs568898116,rs568901867, rs568901932, rs568934389, rs568941411, rs568942492,rs568946298, rs568957792, rs568960972, rs568962977, rs568991081,rs569002664, rs569004282, rs569008496, rs569008981, rs569018043,rs569021977, rs569037344, rs569040125, rs569043543, rs569044282,rs569058233, rs569060347, rs569073744, rs569077509, rs569084649,rs569088134, rs569090666, rs569097375, rs569097717, rs569110116,rs569111463, rs569111906, rs569117797, rs569120336, rs569124635,rs569126067, rs569130123, rs569137113, rs569138368, rs569145095,rs569166731, rs569174432, rs569178705, rs569179920, rs569192205,rs569193701, rs569199948, rs569203916, rs569211552, rs569216242,rs569218319, rs569223254, rs569226249, rs569240925, rs569243124,rs569243581, rs569247264, rs569250547, rs569256702, rs569266170,rs569266470, rs569282076, rs569287779, rs569292665, rs569292686,rs569294733, rs569296160, rs569299436, rs569305111, rs569318098,rs569322483, rs569323574, rs569332424, rs569335856, rs569346184,rs569348395, rs569353425, rs569363364, rs569372045, rs569373153,rs569373676, rs569380208, rs569396354, rs569397163, rs569403069,rs566954577, rs566959426, rs566962640, rs566965232, rs566971958,rs566975411, rs566978352, rs566995047, rs567004279, rs567012978,rs567017170, rs567019497, rs567022849, rs567022972, rs567023573,rs567030519, rs567031257, rs567032101, rs567041197, rs567049320,rs567069923, rs567070563, rs567070914, rs567071205, rs567077490,rs567101741, rs567113616, rs567114804, rs567115734, rs567116280,rs567133889, rs567138494, rs567140729, rs567140891, rs567145819,rs567153009, rs567160469, rs567163638, rs567165169, rs567170082,rs569409403, rs569420680, rs569425049, rs569425476, rs569434159,rs569438603, rs569443008, rs569447890, rs569458248, rs569459150,rs569467514, rs569469533, rs569474091, rs569476290, rs569479148,rs569500103, rs569513195, rs569518947, rs569520274, rs569525109,rs569528827, rs569530987, rs569531100, rs569540833, rs569542827,rs567426042, rs567432854, rs567441455, rs567445908, rs567452449,rs567458795, rs567473198, rs567474262, rs567483099, rs567490004,rs567496436, rs567500163, rs567511147, rs567517434, rs567525312,rs567532840, rs567537049, rs567542887, rs567551084, rs567559518,rs567567781, rs567577812, rs567583950, rs567593721, rs567603268,rs567613785, rs567616296, rs567626121, rs567628506, rs567628553,rs567634404, rs567640274, rs567644060, rs567645794, rs567648352,rs567651816, rs567653530, rs567655144, rs567659849, rs567661274,rs567669994, rs567673049, rs567677231, rs567682871, rs567687581,rs567689705, rs567692831, rs567700281, rs567701451, rs567706815,rs567729103, rs567729394, rs567732660, rs567735088, rs567744279,rs567744352, rs567759416, rs567766358, rs567770003, rs567770613,rs567774865, rs567790981, rs567792083, rs567802797, rs567814427,rs567821188, rs567848030, rs567851309, rs567853705, rs567855999,rs567858366, rs567858750, rs567861184, rs567862721, rs567871541,rs567878525, rs567884441, rs567889091, rs567892603, rs567894819,rs567916236, rs567930433, rs567937306, rs571667304, rs567939090,rs567943104, rs567944439, rs567948971, rs567950706, rs567955258,rs567993123, rs568001081, rs568008158, rs568015048, rs568015745,rs568019123, rs568022429, rs568022848, rs568028034, rs568032950,rs568046777, rs568054228, rs568063202, rs568065710, rs568066813,rs568069582, rs568083172, rs568087570, rs568090984, rs568101668,rs568101885, rs568106435, rs568108719, rs568115633, rs568123248,rs568135181, rs568194140, rs568209629, rs568215108, rs568217807,rs568225980, rs568242913, rs568248458, rs568248692, rs568249694,rs568254410, rs568264651, rs568266157, rs568271264, rs568277386,rs568287760, rs568290584, rs568290986, rs568299380, rs568303881,rs568308729, rs568310591, rs568313914, rs568326151, rs568330395,rs568331607, rs568333092, rs568344965, rs568347944, rs568351685,rs568352187, rs568355329, rs568365845, rs568370851, rs568371319,rs568379012, rs568381633, rs568383170, rs568387960, rs568405025,rs569613241, rs569616197, rs569628018, rs569628392, rs569639298,rs569645219, rs569647303, rs569652952, rs569657056, rs569661529,rs569661553, rs569664112, rs569677565, rs569682557, rs569696433,rs569719409, rs569725227, rs569725472, rs569747017, rs569762219,rs569762509, rs569764056, rs569768179, rs569773614, rs569785753,rs569787001, rs569787634, rs569791421, rs569798380, rs569806129,rs569819283, rs569820423, rs569824072, rs569839327, rs569849647,rs569849677, rs569861169, rs569879060, rs569900432, rs569901441,rs569902273, rs569915379, rs569916070, rs569916494, rs569918595,rs569918892, rs569923207, rs569931921, rs569940428, rs569948840,rs569954221, rs569955923, rs569959186, rs569963627, rs569964422,rs569966497, rs569974196, rs569975645, rs569976531, rs569976800,rs569986168, rs569986188, rs569987785, rs569990437, rs569992628,rs569995074, rs569998961, rs570002252, rs570007601, rs570009435,rs570026522, rs570033137, rs570034030, rs570037623, rs570040930,rs570048876, rs570060556, rs570066905, rs570076402, rs570083778,rs570094447, rs570094850, rs570104654, rs570116054, rs570116422,rs570121717, rs570123112, rs570146554, rs570147964, rs570154693,rs570155129, rs570158813, rs570170484, rs570174330, rs570181273,rs570183926, rs570197199, rs570200458, rs570202257, rs570240395,rs570245744, rs570257635, rs570259574, rs570266601, rs570273930,rs570290680, rs570292264, rs570298813, rs570419159, rs570425143,rs570432981, rs570439409, rs570442395, rs570450465, rs570455595,rs570457737, rs570461635, rs570469256, rs570472486, rs570477298,rs570482555, rs570487228, rs570510873, rs570511221, rs570514935,rs570516403, rs570521267, rs570535658, rs570536203, rs570536545,rs570542674, rs570555945, rs570556723, rs570559278, rs570573683,rs570578757, rs570582067, rs570587998, rs570588226, rs570605079,rs570612007, rs570617581, rs570621162, rs570625442, rs570630346,rs570632156, rs570641256, rs570646397, rs570652136, rs570663490,rs570665940, rs570678488, rs570681217, rs570685375, rs570696828,rs570697271, rs570706429, rs570707990, rs570714386, rs570715071,rs570717703, rs570720089, rs570728993, rs570730941, rs570732992,rs570738769, rs570739145, rs570747793, rs570768449, rs570769647,rs570777929, rs570787518, rs570789708, rs570800455, rs570802053,rs570808320, rs570820333, rs570822413, rs570826682, rs570826912,rs570849603, rs570852075, rs570856882, rs570858965, rs570868303,rs570870376, rs570873557, rs570891540, rs570898542, rs570929000,rs570934000, rs570943684, rs570950736, rs570983343, rs570984340,rs570987705, rs570993245, rs570993643, rs570993658, rs570995259,rs570997302, rs571001053, rs571024716, rs571026949, rs571029819,rs571036471, rs571051971, rs571071751, rs571084371, rs571097630,rs571104207, rs571104431, rs571105904, rs571107281, rs571110951,rs571112309, rs571116965, rs571133256, rs569548410, rs569548952,rs569554673, rs569558850, rs571144342, rs571145614, rs571161190,rs571163838, rs571169169, rs571172017, rs571176982, rs571177134,rs571178041, rs571180911, rs571183732, rs571207590, rs571208268,rs571211156, rs571211530, rs571217129, rs571218388, rs571227005,rs571229541, rs571238563, rs571239816, rs571242336, rs571244186,rs571256482, rs571270274, rs571270997, rs571271730, rs571275931,rs571276662, rs571292304, rs571296152, rs571298774, rs571299980,rs571312303, rs571347011, rs571347293, rs571348736, rs571349384,rs571360399, rs571360899, rs571380198, rs571389880, rs571393942,rs571394460, rs571394931, rs571411087, rs571419238, rs571419923,rs571421248, rs571425591, rs571436182, rs571444181, rs571452294,rs571454971, rs571456103, rs571460662, rs571467135, rs571525981,rs571535905, rs571541355, rs571545999, rs571548747, rs571554543,rs571560986, rs571561266, rs571562611, rs571573456, rs571574736,rs571585394, rs571600356, rs571608246, rs571623566, rs571623786,rs571625080, rs571629868, rs571636659, rs571641933, rs571645057,rs571649019, rs571653397, rs571654469, rs571657309, rs571675467,rs571678067, rs571688196, rs571690687, rs571699037, rs571709996,rs571713091, rs571713622, rs571716801, rs571724231, rs571726863,rs571741147, rs571742188, rs571749360, rs571753711, rs571772795,rs571783573, rs571784546, rs569569587, rs569572350, rs569572409,rs569581277, rs571789377, rs571800585, rs571801009, rs571805020,rs571809850, rs571810380, rs571815735, rs571819584, rs571821312,rs569583259, rs569588373, rs569594237, rs574065511, rs572074695,rs572076543, rs572084100, rs572085642, rs572112127, rs572121185,rs572126830, rs572131760, rs572145739, rs572152580, rs572152647,rs572160442, rs572164097, rs572165657, rs572167859, rs572175276,rs572181263, rs572186973, rs572189775, rs572190560, rs572199411,rs572222807, rs572226485, rs572238666, rs572238789, rs572239622,rs572242342, rs572246450, rs572247779, rs572252893, rs572255880,rs572259461, rs572263364, rs572265552, rs572265570, rs572275896,rs572276371, rs572283815, rs572285395, rs572285891, rs572290906,rs572291170, rs572305413, rs572306525, rs572324097, rs572324737,rs572350279, rs572352489, rs572353028, rs572358844, rs572359512,rs572363891, rs572366988, rs572377776, rs572382957, rs572383359,rs572384219, rs572397888, rs572400216, rs572400464, rs572402281,rs572407797, rs572409277, rs572412289, rs572414358, rs572416203,rs572423840, rs572425500, rs572433972, rs572444179, rs572465982,rs572468602, rs572480123, rs572480133, rs572480409, rs572480556,rs572481090, rs572485223, rs572485823, rs572489975, rs572499406,rs572508554, rs572512115, rs572518425, rs572527285, rs572528186,rs572541406, rs572557006, rs572560307, rs572563046, rs572567511,rs572570267, rs572577111, rs572589048, rs572590956, rs572592255,rs572596877, rs572596906, rs572598455, rs572616878, rs572617036,rs572618565, rs572619191, rs572621436, rs572622399, rs572630047,rs572634907, rs572638046, rs570320661, rs570325582, rs570326623,rs570335522, rs570341586, rs570344991, rs570365274, rs570368836,rs570377103, rs570381147, rs570391677, rs570392298, rs570393629,rs570394906, rs570401883, rs570402887, rs570408842, rs572645712,rs572645998, rs572652405, rs572654525, rs572655305, rs572673729,rs572675684, rs572676391, rs572676837, rs572698760, rs572707016,rs572709319, rs572712366, rs572728566, rs572736727, rs572737942,rs572741695, rs572763715, rs572765864, rs572768414, rs572771881,rs572773600, rs572777891, rs572797113, rs572802012, rs572805029,rs572806470, rs572807520, rs572808126, rs572809013, rs572812426,rs572820932, rs572825410, rs572828359, rs572835419, rs572836146,rs572837898, rs572846647, rs572851741, rs572857542, rs573031593,rs573033617, rs573065815, rs573071976, rs573073808, rs573078206,rs573080980, rs573084783, rs573089555, rs573090581, rs573097972,rs573102052, rs573104634, rs573104783, rs573104821, rs573125334,rs573135171, rs573136111, rs573140514, rs573140676, rs573143610,rs573149898, rs573152149, rs573153211, rs573159033, rs573160236,rs573180350, rs573204855, rs573208323, rs573212837, rs573215058,rs573223461, rs573232241, rs573232569, rs573235970, rs573239459,rs573245020, rs573245556, rs573268753, rs573287053, rs573289475,rs573294668, rs573299865, rs573317691, rs573318092, rs573319052,rs573327165, rs573344944, rs573354996, rs573361326, rs573370564,rs573371188, rs573382059, rs573384755, rs573395796, rs573404827,rs573411407, rs573416367, rs573425220, rs573427467, rs573436588,rs573442915, rs573444141, rs573444150, rs573450126, rs573452178,rs573458624, rs573461476, rs573462049, rs573465244, rs573466003,rs573467369, rs573471128, rs573492228, rs573493216, rs573498635,rs573507228, rs573509656, rs573529526, rs573538798, rs573555425,rs573558299, rs573558966, rs573562370, rs573562651, rs573562897,rs573572961, rs573574213, rs573584355, rs573599137, rs573604472,rs573631845, rs573631860, rs573632773, rs573636945, rs573639615,rs573643862, rs573644489, rs573644816, rs573660306, rs573665178,rs573671648, rs573673215, rs573677583, rs573687511, rs573691261,rs573695130, rs573695572, rs573702608, rs573706666, rs573717835,rs573721741, rs573726350, rs573732040, rs573732150, rs573733730,rs573748655, rs573750733, rs573781916, rs573783542, rs573788865,rs573790618, rs573798324, rs573801479, rs573809979, rs573818351,rs573826759, rs573841371, rs573844696, rs573847027, rs573848788,rs573851219, rs573851954, rs573855640, rs573857932, rs573867201,rs573871810, rs573881602, rs573885970, rs573896240, rs573896801,rs573902173, rs573906372, rs573909284, rs573934153, rs573950818,rs573951078, rs573970444, rs573972330, rs573973034, rs573995295,rs573996724, rs574003315, rs574005067, rs574006425, rs574008354,rs574010024, rs574014670, rs574017741, rs574020748, rs574027763,rs574042507, rs574055132, rs574060906, rs571838406, rs571838799,rs571868051, rs571869666, rs571871118, rs571876768, rs571886294,rs571890894, rs571894343, rs571905360, rs571911853, rs571913216,rs571923622, rs571930867, rs571932810, rs571933551, rs571935623,rs571941738, rs571947079, rs571952038, rs571953507, rs571959958,rs571962635, rs571963335, rs571969658, rs571980266, rs571983477,rs571984084, rs571985536, rs571987106, rs571997585, rs571999828,rs572002102, rs572010686, rs572011710, rs572015212, rs572021434,rs572032580, rs572040340, rs572040841, rs572044158, rs572045228,rs572048506, rs572056393, rs572071576, rs574062516, rs574064898,rs574083877, rs574087302, rs574109905, rs574134206, rs574138316,rs574148793, rs574157517, rs574163911, rs574165397, rs574165573,rs574169496, rs574172713, rs574173898, rs574179318, rs574183494,rs574189151, rs574192681, rs574196162, rs574198801, rs574208450,rs574210051, rs574216094, rs574223948, rs574226470, rs574226480,rs574237901, rs574250492, rs574263914, rs574269081, rs574274345,rs574274699, rs574277093, rs574285810, rs574293316, rs574295508,rs574303016, rs574304451, rs574311375, rs574312675, rs574313950,rs574315368, rs574315990, rs574318532, rs574347175, rs574347942,rs574358257, rs574365117, rs574388443, rs574400298, rs574404852,rs574425756, rs574431136, rs574432772, rs574438151, rs574442178,rs574445331, rs574453342, rs574464133, rs574465254, rs574467301,rs574486843, rs574486971, rs574494304, rs574510232, rs574511419,rs576857055, rs576857815, rs574516099, rs574525211, rs574538055,rs574541052, rs574542125, rs574553576, rs574554298, rs574556955,rs574563913, rs574597246, rs574600362, rs574616776, rs574617303,rs574620669, rs574624099, rs574629162, rs574632218, rs574643342,rs574651148, rs574652619, rs574653959, rs574656518, rs574671427,rs574686832, rs577016092, rs574704378, rs574728889, rs574737182,rs574756260, rs574768206, rs574772013, rs574777998, rs574802468,rs574804367, rs574815882, rs574825001, rs574826954, rs574827221,rs574828964, rs574837116, rs574839245, rs574844754, rs574858257,rs574859194, rs574861621, rs574862888, rs574863120, rs574866055,rs574868756, rs574870819, rs574901182, rs574901435, rs574908726,rs574908861, rs574912645, rs574913771, rs574938896, rs574942848,rs574947263, rs574951540, rs574952240, rs574971282, rs574988489,rs575000136, rs575019696, rs575025479, rs575030700, rs575033706,rs575039690, rs575039991, rs575046731, rs575049808, rs575052360,rs575058843, rs575064239, rs575070688, rs575071022, 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rs575856576,rs575856595, rs575860620, rs575861031, rs575874083, rs575874331,rs575874514, rs575876716, rs575889965, rs575898246, rs575899340,rs575906624, rs575909799, rs575928739, rs575938092, rs575948642,rs575974406, rs575978744, rs575980146, rs575981668, rs576004410,rs576006665, rs576012946, rs576013428, rs576075866, rs576077242,rs576080286, rs576092833, rs576094173, rs576098997, rs576099613,rs576108721, rs576116132, rs576129632, rs576133345, rs576137521,rs576142856, rs576143112, rs576199358, rs576203402, rs576204417,rs576218978, rs576220247, rs576222371, rs576231932, rs576232513,rs576233805, rs576235440, rs576241036, rs576248457, rs576248701,rs576275614, rs576276604, rs576292956, rs576295570, rs574065890,rs576459191, rs745731253, rs576462629, rs576463690, rs576470101,rs576476285, rs576483102, rs576493937, rs576501198, rs576505179,rs576506127, rs576522998, rs576524718, rs576535407, rs576538812,rs576539707, rs576544278, rs576546750, rs576548321, rs576560023,rs576564875, rs576576942, 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rs576064444, rs576070871, rs745556963,rs745561554, rs745576280, rs745595519, rs745603347, rs745606351,rs745609211, rs745610733, rs745612731, rs745615387, rs745622180,rs745625660, rs745627193, rs745630626, rs576152502, rs576157606,rs576158565, rs576158589, rs576162921, rs576165797, rs576170336,rs576171998, rs576176699, rs576177449, rs576188597, rs576189016,rs576194266, rs576197624, rs745631208, rs745638890, rs745642619,rs745654067, rs745655787, rs745668571, rs576312090, rs576319108,rs576326302, rs576326964, rs576332097, rs576336022, rs576340918,rs576351738, rs576358640, rs576366276, rs576371996, rs576374568,rs576393821, rs576399375, rs576404073, rs576417295, rs576419889,rs576420957, rs576421888, rs576423504, rs576432865, rs576432976,rs576437093, rs576451709, rs576455645, rs745822652, rs745826981,rs745836378, rs745844054, rs745853378, rs745856272, rs745860223,rs745861252, rs745864115, rs745865858, rs745869876, rs745873888,rs745882475, rs745900195, rs745901600, rs745904855, rs745919709,rs745920974, rs745927017, rs745937531, rs745939371, rs745954944,rs748534826, rs745957268, rs745958606, rs745973441, rs745973937,rs745980267, rs745982226, rs746012797, rs746018040, rs746018253,rs746025528, rs746030193, rs746050528, rs746051909, rs746053244,rs746053577, rs746060361, rs746071460, rs746077267, rs746082665,rs746089098, rs746089439, rs746093844, rs746096101, rs746098125,rs746098799, rs746102402, rs746111812, rs746142471, rs746145094,rs746145216, rs746162818, rs746168914, rs746170985, rs746171962,rs746176302, rs746177547, rs746182950, rs746190862, rs746197138,rs746199492, rs746208375, rs746215144, rs746223144, rs746231972,rs746241982, rs746244583, rs746245369, rs746252547, rs746267892,rs746278258, rs746296195, rs746300163, rs746304887, rs746308619,rs746312645, rs746314195, rs746320328, rs746329368, rs746352083,rs746358373, rs746358573, rs746359201, rs746362347, rs746364377,rs746368252, rs746371925, rs746382874, rs746386179, rs746396716,rs746398636, rs746408577, rs746408905, rs746410351, rs746413884,rs746419982, rs746420867, rs746423885, rs746424525, rs746435794,rs746445432, rs746447922, rs746450127, rs746461881, rs746463571,rs746465133, rs746474737, rs746478123, rs746480921, rs746489667,rs746496447, rs746504959, rs746507786, rs746509122, rs746517252,rs746522372, rs746530678, rs746533060, rs746542678, rs746544784,rs746549216, rs746549944, rs746552830, rs746558116, rs746567382,rs746580087, rs746595383, rs746595475, rs746608555, rs746610120,rs746621707, rs746629482, rs746631494, rs746636576, rs746650607,rs746676585, rs746686539, rs746692871, rs746703326, rs746705219,rs746715201, rs746717153, rs746721075, rs746728602, rs746732807,rs746734022, rs746751630, rs746754796, rs746755248, rs746757578,rs746761896, rs746772180, rs746782713, rs746786647, rs746799882,rs746800041, rs746803134, rs746803774, rs746815971, rs746824464,rs746825484, rs746826904, rs746843087, rs746848356, rs746852797,rs746854571, rs746860346, rs746878330, rs746891368, rs746903630,rs746919900, rs746922672, rs746922960, rs746924679, rs746928991,rs746940075, rs746951702, rs746954465, rs746957636, rs746959710,rs746960119, rs746960342, rs746961845, rs746987860, rs746989537,rs746993452, rs746999256, rs747000990, rs747007238, rs747010020,rs747023706, rs747027713, rs747031049, rs747039315, rs747054322,rs747055164, rs747056776, rs747062451, rs747063453, rs747066651,rs747068584, rs747073304, rs747093507, rs747097095, rs747120921,rs747139833, rs747155601, rs747165943, rs747167478, rs747172083,rs747174075, rs747197438, rs747198374, rs747216152, rs747226736,rs747241571, rs747246785, rs747248485, rs747257293, rs747259952,rs747261526, rs747268674, rs747289352, rs747298108, rs747298334,rs747309150, rs747313817, rs747317307, rs747335580, rs747340486,rs747351741, rs747355388, rs747379692, rs747397469, rs747404264,rs747410020, rs747435443, rs747442039, rs747449396, rs747463339,rs747470665, rs747475842, rs747493174, rs747496733, rs747497177,rs747501648, rs747503680, rs747504216, rs747506334, rs747513010,rs747518953, rs747524732, rs747527098, rs747532231, rs747548904,rs747551854, rs747552815, rs747557701, rs747563674, rs747567594,rs747570518, rs747576695, rs747584023, rs747590302, rs747594490,rs747600984, rs747601518, rs747611379, rs747625579, rs747633658,rs747646690, rs747653654, rs747663585, rs747664134, rs747668487,rs747671624, rs747685922, rs747686883, rs747688625, rs747691476,rs747699590, rs747701130, rs747721717, rs747743339, rs747746739,rs747750560, rs747764340, rs747765479, rs747779647, rs747797791,rs747800397, rs747807379, rs747810788, rs747826656, rs747826969,rs747842197, rs747847315, rs747849396, rs747858955, rs747883459,rs747884179, rs747888193, rs747893750, rs747898251, rs747913469,rs747922493, rs747934209, rs747950128, rs747957147, rs747961978,rs747962334, rs747971405, rs747998176, rs748032043, rs748037584,rs748060270, rs748065488, rs748067255, rs748069143, rs748073264,rs748075132, rs748075212, rs748080458, rs748086596, rs748087010,rs748093597, rs748096011, rs748099580, rs748104056, rs748110380,rs748117093, rs748122817, rs748128175, rs748131207, rs748136696,rs748138574, rs748153935, rs748154640, rs748155272, rs748170147,rs748176768, rs748181734, rs748197708, rs748199778, rs748204315,rs748209011, rs748210347, rs748214290, rs748239755, rs748240735,rs748241978, rs748247426, rs745672172, rs745672897, rs745674292,rs745691380, rs745694341, rs745697624, rs745698046, rs745702613,rs745705632, rs745710217, rs745723485, rs748250889, rs748258775,rs748263897, rs748266161, rs748270680, rs748279830, rs748292110,rs748311113, rs745730332, rs745744527, rs745765360, rs745776167,rs745786458, rs745792735, rs745793146, rs745793522, rs745795953,rs745814195, rs745820634, rs748389384, rs748405719, rs748410208,rs748436826, rs748437585, rs748440003, rs748441509, rs748444002,rs748459329, rs748460702, rs748462120, rs748467636, rs748476040,rs748478232, rs748483109, rs748485447, rs748508795, rs748513502,rs748513613, rs748518228, rs748531061, rs748535946, rs748537007,rs748542623, rs748550940, rs748569994, rs748577376, rs748578698,rs748578752, rs748580686, rs748591752, rs748593161, rs748595599,rs748598196, rs748612994, rs748637172, rs748640826, rs748641331,rs748643063, rs748646994, rs748648532, rs748651563, rs748652575,rs748653463, rs748656767, rs748661007, rs748661808, rs748680741,rs748684564, rs748692688, rs748701963, rs748977815, rs748980605,rs748985832, rs749006644, rs749009396, rs749009596, rs749009907,rs749021169, rs749022165, rs749030087, rs749036377, rs749037576,rs749040329, rs751303262, rs751311537, rs751314870, rs751315639,rs751319659, rs751320836, rs751348608, rs749043092, rs749049807,rs749066489, rs749068030, rs749070715, rs749100610, rs749107855,rs749134007, rs749157742, rs749158704, rs749160219, rs749169226,rs749170659, rs749176060, rs749188432, rs749196767, rs749205997,rs749207896, rs749208075, rs749211754, rs751479037, rs751479518,rs751482754, rs749215347, rs749220173, rs749221295, rs749228182,rs749229696, rs749237133, rs749248494, rs749254120, rs749254755,rs749257073, rs749259188, rs749262644, rs749275110, rs749288798,rs749291870, rs749294037, rs749295557, rs749307242, rs749309929,rs749315987, rs749318223, rs749318240, rs749319166, rs749333466,rs749340840, rs749343358, rs749345113, rs749348831, rs749352734,rs749354113, rs749358286, rs749367272, rs749389287, rs749392753,rs749396029, rs749398851, rs749409711, rs749419247, rs749445449,rs749450157, rs749455875, rs749458334, rs749470287, rs749485204,rs749499641, rs749503690, rs749503696, rs749503953, rs749507197,rs749509095, rs749521010, rs749534218, rs749536239, rs749547569,rs749547812, rs749556026, rs749556457, rs749558108, rs749563862,rs749566174, rs749579344, rs749589510, rs749589894, rs749597818,rs749602006, rs749608812, rs749609849, rs749618933, rs749622750,rs749622820, rs749628867, rs749634553, rs749637474, rs749638304,rs749653326, rs749654346, rs749655325, rs749664538, rs749679350,rs749682121, rs749684027, rs749684330, rs749684607, rs749688835,rs749723831, rs749724040, rs749727911, rs749728290, rs749728629,rs749734187, rs749738695, rs749739794, rs749752831, rs749753523,rs749756006, rs749769666, rs749774960, rs749778466, rs749783600,rs749790411, rs749795504, rs749801428, rs749802042, rs749816348,rs749822211, rs749830625, rs749834574, rs749842400, rs749853614,rs749861799, rs749863186, rs749867719, rs749872352, rs749876921,rs749878100, rs749883554, rs749891518, rs749892539, rs749906002,rs749909456, rs749909655, rs749920625, rs749929391, rs749929397,rs749934264, rs749940389, rs749948427, rs749956529, rs749966912,rs749967523, rs749982142, rs749985665, rs749985779, rs749985969,rs749993062, rs749993575, rs750001424, rs750011855, rs750011877,rs750018186, rs750019417, rs750022865, rs750023540, rs750024053,rs750027601, rs750033025, rs750037616, rs750049971, rs750060761,rs750071139, rs750074093, rs750079287, rs750092828, rs750097818,rs750108753, rs750120872, rs750122332, rs750135009, rs750141817,rs750150515, rs750155140, rs750156297, rs750156301, rs750174159,rs750176346, rs750180591, rs750183921, rs750193044, rs748314030,rs748314631, rs748319164, rs748322174, rs748322181, rs748322678,rs748323212, rs748327701, rs748337129, rs748339164, rs748342303,rs748350184, rs748350303, rs748356638, rs748366812, rs748386175,rs748386641, rs750745177, rs752682709, rs750760171, rs750762427,rs750765053, rs750770301, rs750771147, rs750778436, rs750779311,rs750793562, rs750810985, rs750840918, rs750857084, rs750860038,rs750861606, rs750869952, rs750870994, rs750872676, rs750873004,rs750878970, rs750879443, rs750897936, rs750905524, rs750909654,rs750915581, rs750925301, rs750949519, rs750953185, rs750965634,rs750992785, rs750993494, rs750995659, rs750996298, rs750998843,rs750999165, rs751035990, rs751037519, rs751038329, rs751039262,rs751056095, rs751059913, rs751060204, rs751072159, rs751078798,rs751100266, rs751102611, rs751102866, rs751119598, rs751125349,rs751132194, rs751134311, rs751147602, rs751150491, rs751155316,rs751157518, rs751160608, rs751170586, rs751189715, rs751190205,rs751197411, rs751210663, rs751225779, rs748705185, rs748706648,rs748717826, rs748721017, rs748725633, rs748739614, rs748745043,rs748749525, rs748749631, rs748754290, rs748755270, rs748755827,rs748779007, rs748784773, rs748795801, rs748808195, rs748820272,rs748831865, rs748844851, rs748857851, rs748876878, rs748878359,rs748891355, rs748897938, rs748906373, rs748909560, rs748925371,rs748929817, rs748936097, rs748936659, rs748947529, rs748950220,rs748966295, rs748966374, rs748967305, rs748971171, rs748971832,rs748977510, rs751227230, rs751231200, rs751232956, rs751236724,rs751245552, rs751248222, rs751267412, rs751273795, rs751280118,rs751287713, rs751291931, rs751296314, rs751353183, rs751355901,rs751369613, rs751377396, rs751383558, rs751409431, rs751420029,rs751421308, rs751422706, rs751423276, rs751424276, rs751431582,rs751435420, rs751444354, rs751450340, rs751463363, 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rs753317816,rs753318380, rs753320880, rs753324021, rs753327649, rs753334791,rs753340329, rs753342503, rs753343664, rs753344703, rs753348642,rs753354398, rs753357724, rs753358549, rs753371914, rs753374951,rs753386006, rs753393380, rs753394110, rs753400215, rs753407114,rs753421846, rs753426169, rs753429281, rs753431411, rs753434043,rs753436390, rs753437904, rs753451598, rs753463469, rs753468802,rs753474558, rs753482913, rs753508660, rs753521422, rs753528363,rs753538742, rs753547983, rs753554618, rs753554708, rs753563510,rs753573896, rs753575090, rs753579095, rs753579657, rs753580807,rs753581263, rs753586466, rs753600710, rs753604319, rs753613105,rs753613869, rs753620554, rs753626269, rs753631404, rs753637694,rs753638845, rs753640612, rs753656796, rs753660077, rs753663353,rs753666936, rs753668709, rs753672638, rs753682192, rs753692752,rs753695966, rs753701241, rs753718025, rs753719936, rs753720222,rs753725061, rs753728395, rs753730995, rs753733974, rs753740248,rs753753174, rs753762133, 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rs766027605, rs766029394, rs766031713,rs766048766, rs766059130, rs766060170, rs763797691, rs763809529,rs763815085, rs763817716, rs763823324, rs766065403, rs766068522,rs766083211, rs766089430, rs766092576, rs766108328, rs766112494,rs766114999, rs766132257, rs766138549, rs766144059, rs766149615,rs766150464, rs766157478, rs761661094, rs761661963, rs761676178,rs761681080, rs761686092, rs761709909, rs761711690, rs761716998,rs761718766, rs761733499, rs761735732, rs761744278, rs761749389,rs761750249, rs761753604, rs766166672, rs766201684, rs766204246,rs766208415, rs766208681, rs766215385, rs766218431, rs766230526,rs766237178, rs766239857, rs766241628, rs766248964, rs766251600,rs766251655, rs766253443, rs766256561, rs766259143, rs766268324,rs766278942, rs766284768, rs766293400, rs766298283, rs766303416,rs766305231, rs766305535, rs766305869, rs766308396, rs766310031,rs766319612, rs764028090, rs764028418, rs764035530, rs764038553,rs764039325, rs764044374, rs764046130, rs764062922, rs764065538,rs764065854, rs764066695, rs764073850, rs764080430, rs764094262,rs764096749, rs764097193, rs764097683, rs764114324, rs764116112,rs764121444, rs764121874, rs764123421, rs764143385, rs764145001,rs764151048, rs764166543, rs764167452, rs764167949, rs764171208,rs764173988, rs764179497, rs764185644, rs764190745, rs764192142,rs764199778, rs764200047, rs764226347, rs764231745, rs764254890,rs764256034, rs764259294, rs764266309, rs764276618, rs764290214,rs764298055, rs764301295, rs764303189, rs764321084, rs764325584,rs764326957, rs764334560, rs764335111, rs764344292, rs764348227,rs764349154, rs764349807, rs764351581, rs764354311, rs764354677,rs764355216, rs764356106, rs764356622, rs764367989, rs764369808,rs764373219, rs764385150, rs764394895, rs764395841, rs764399992,rs764413219, rs764414233, rs764415189, rs764415343, rs764426997,rs764427254, rs764437770, rs764438281, rs764445637, rs764448678,rs764451029, rs764453749, rs764454662, rs764455484, rs764467896,rs764482742, rs764486645, 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rs778529037, rs778535459, rs778544896,rs778549576, rs778551823, rs778562686, rs778571859, rs778575794,rs778579213, rs778579490, rs778585936, rs778602545, rs778607397,rs778608373, rs778629253, rs778637508, rs778646358, rs778665439,rs778667205, rs778671486, rs778672788, rs778677738, rs778680278,rs778681682, rs778684195, rs778691338, rs778694461, rs778718387,rs778720286, rs778724569, rs778732353, rs778756219, rs778779053,rs778781142, rs778782838, rs778783877, rs778784443, rs778789988,rs778826465, rs778826731, rs778829910, rs778834718, rs778841994,rs778846264, rs778853347, rs778855194, rs778863313, rs778864395,rs778868324, rs778868899, rs778872942, rs778876657, rs778895924,rs778902389, rs778910995, rs778912605, rs778921788, rs778924212,rs778935150, rs778959463, rs778961042, rs778961601, rs778970510,rs778977003, rs778977579, rs778992667, rs778997949, rs779004400,rs779013146, rs779017029, rs779021570, rs779023109, rs779023513,rs779023990, rs779032368, rs779038935, rs779039134, rs779044617,rs780953843, rs779047450, rs779064972, rs779065539, rs779067444,rs779086046, rs779086652, rs779093024, rs779100071, rs779107280,rs779110809, rs779115007, rs779120667, rs779124168, rs779130886,rs779131078, rs779132706, rs779162179, rs779170808, rs779174665,rs779175890, rs779179467, rs779183288, rs779194241, rs779195155,rs779197941, rs779198752, rs779199360, rs779208517, rs779211610,rs779211837, rs779217558, rs779218285, rs779232345, rs779238791,rs779242097, rs779265904, rs779274617, rs779275480, rs779279807,rs779290728, rs779293842, rs779297819, rs779334556, rs779335927,rs779342598, rs779344276, rs779347555, rs779366573, rs779367315,rs779367548, rs779374125, rs779377691, rs779380535, rs779399222,rs779404027, rs779409039, rs779411921, rs779418922, rs779443197,rs779447320, rs779452931, rs779458918, rs779462358, rs779465426,rs779469814, rs781352542, rs779481562, rs779513822, rs779521780,rs779525872, rs779529332, rs779533447, rs779543061, rs779544589,rs779552241, rs779553271, rs779556027, rs779565066, rs779565682,rs779577646, rs779579119, rs779581098, rs779597157, rs779598000,rs779601519, rs779610153, rs779613170, rs779614842, rs779614920,rs779616185, rs779618537, rs779621177, rs779631878, rs777357296,rs777363270, rs777376467, rs777387737, rs777397292, rs777397650,rs777399547, rs777409809, rs777415055, rs777421215, rs777424872,rs777442552, rs777448577, rs777453672, rs777456245, rs777473225,rs777474980, rs777478112, rs777485724, rs777489460, rs777491499,rs777497719, rs777498324, rs777511903, rs777517698, rs777519211,rs777522761, rs777524146, rs777525718, rs777525955, rs777531367,rs779632837, rs779634266, rs779643384, rs779651784, rs779655565,rs779659528, rs779660666, rs777534182, rs777547710, rs777562023,rs777563411, rs777567997, rs777568665, rs777569252, rs777580111,rs777585237, rs777586553, rs777597187, rs777597943, rs777602651,rs777610503, rs777612124, rs777631328, rs777642235, rs777645089,rs777645723, rs777648846, rs777652556, rs777656930, rs777659083,rs777672724, rs777679288, rs777680752, rs777685728, rs779684023,rs779688780, rs779701833, rs779713171, rs779719624, rs779720077,rs779720268, rs779730124, rs779743126, rs779746251, rs779749486,rs779760566, rs777688542, rs777689934, rs777696275, rs777699187,rs777701009, rs777704923, rs777711276, rs777714401, rs777719997,rs777723842, rs777745382, rs777752117, rs777759134, rs777763160,rs777786864, rs777794928, rs777796919, rs779779837, rs779789655,rs779797030, rs779801263, rs779810430, rs779816550, rs779896869,rs779905165, rs779909389, rs779909937, rs779910289, rs779912212,rs779913494, rs779918212, rs779935228, rs779940433, rs779942107,rs779946839, rs779955729, rs779962549, rs779966634, rs779977328,rs779980156, rs779983411, rs779992902, rs780021093, rs780024160,rs780025981, rs780032369, rs780033023, rs780036041, rs780036244,rs780044095, rs780048451, rs780054613, rs780070933, rs780081433,rs780088249, rs780093368, rs780097005, rs780112712, rs780129218,rs780136244, rs780143514, rs780150460, rs780161870, rs780173415,rs780184990, rs780185352, rs780191288, rs780192954, rs780193075,rs780204534, rs780209843, rs780213591, rs780223003, rs780224606,rs780226161, rs780229894, rs780247806, rs780250744, rs780252368,rs780258680, rs780271241, rs780272417, rs778199167, rs778200981,rs778205582, rs778212700, rs778224132, rs778227050, rs778234057,rs778245111, rs778256583, rs778264203, rs778267744, rs778267919,rs778268072, rs778273142, rs778274803, rs778280535, rs778286455,rs778286555, rs778287806, rs780273168, rs780277097, rs780284561,rs780286452, rs780287717, rs780295014, rs780298600, rs780302019,rs780306874, rs780309552, rs780311091, rs780321147, rs780323436,rs780328810, rs780334364, rs780339867, rs780354499, rs780357870,rs780360039, rs780365231, rs780367768, rs780373373, rs780377126,rs780382019, rs780385127, rs780415042, rs780428553, rs780429920,rs780430588, rs780431573, rs780440299, rs780459247, rs780464396,rs780467949, rs780470144, rs780482988, rs780489765, rs780499008,rs780503439, rs780506373, rs780511645, rs780521198, rs780538937,rs780539607, rs780543953, rs780545824, rs780548068, rs780548556,rs780549091, rs780552428, rs780552753, rs780561045, rs780561330,rs780568466, rs780569360, rs780572490, rs780581436, rs780584237,rs780588097, rs780590460, rs780604177, rs780606972, rs780614013,rs780616532, rs780627705, rs780629749, rs780631368, rs780635987,rs780639633, rs780654141, rs780664100, rs780664734, rs780667555,rs780668568, rs780668904, rs780670645, rs780675052, rs780677565,rs780679795, rs780687266, rs780688987, rs780692722, rs780694641,rs780696776, rs780699045, rs780699490, rs780702892, rs780709709,rs780714233, rs780716059, rs780721698, rs780735752, rs780736047,rs780748005, rs780752912, rs780773579, rs780776903, rs780777406,rs780791780, rs780794669, rs780800610, rs780833660, rs780854715,rs780860595, rs780863489, rs780866374, rs780868780, rs780869902,rs780883837, rs780886899, rs780889674, rs780896979, rs780907653,rs780911865, rs780916622, rs780929270, rs780933574, rs780934443,rs780937710, rs780947179, rs780957178, rs780978248, rs780980939,rs780984808, rs780985908, rs780998645, rs781002177, rs781002194,rs781005655, rs781013450, rs781019079, rs781026477, rs781029812,rs781030866, rs781050705, rs781060448, rs781061507, rs781073509,rs781078047, rs781097173, rs781103753, rs781125032, rs781130334,rs781131002, rs781136021, rs781137722, rs781142612, rs781147912,rs781155719, rs781158421, rs781164409, rs781173502, rs781177056,rs781182824, rs781187420, rs781196136, rs781205539, rs781209555,rs781211921, rs781217313, rs781218182, rs781232065, rs781235719,rs781239330, rs781248562, rs781249043, rs781250079, rs781252013,rs781259148, rs781276186, rs781283847, rs781284349, rs781285931,rs781290434, rs781297445, rs781297648, rs781302215, rs781302360,rs781303126, rs781314927, rs781324036, rs781324180, rs781334557,rs781343837, rs781349157, rs781354333, rs781358976, rs781380775,rs781383506, rs781388224, rs781393483, rs781394568, rs781398089,rs781404700, rs781407915, rs781418996, rs781419696, rs781429620,rs781443247, rs781443469, rs781447654, rs781451713, rs781453448,rs781465865, rs781466747, rs781470200, rs781494779, rs781495640,rs781508964, rs781514617, rs781520828, rs781526626, rs781549032,rs781552044, rs781553326, rs781567063, rs781571097, rs781583938,rs781584137, rs781586703, rs781590903, rs781595930, rs781615405,rs781617373, rs781618404, rs781678785, rs781683854, rs781684931,rs781688016, rs781694497, rs781695215, rs781696286, rs781702897,rs781706542, rs781716248, rs781718704, rs781739425, rs781746467,rs781779248, rs781779990, rs781780889, rs781781140, rs779831834,rs779833684, rs779835722, rs779840795, rs779842414, rs779853811,rs779855299, rs779858767, rs779888760, rs781634179, rs781636306,rs781638032, rs781644101, rs781649313, rs781649376, rs781654169,rs781662571, rs781669534, rs781669618, and rs781671404.

In one example, the guide RNA used in the present disclosure maycomprise at least one 20 nucleotide (nt) target nucleic acid sequencelisted in Table 5. Provided in Table 5 are the gene symbol and thesequence identifier of the gene (Gene SEQ ID NO), the gene sequenceincluding 1-5 kilobase pairs upstream and/or downstream of the targetgene (Extended Gene SEQ ID NO), and the 20 nt target nucleic acidsequence (20 nt Target Sequence SEQ ID NO). In the sequence listing therespective target gene, the strand for targeting the gene (noted by a(+) strand or (−) strand in the sequence listing), the associated PAMtype and the PAM sequence are described for each of the 20 nt targetnucleic acid sequences (SEQ ID NO: 5305-312,293). It is understood inthe art that the spacer sequence, where “T” is “U,” may be an RNAsequence corresponding to the 20 nt sequences listed in Table 5.

TABLE 5 Nucleic Acid Sequences Extended 20 nt Target Gene Gene SequenceGene SEQ SEQ SEQ Symbol ID NO ID NO ID NO ATXN1 5303 5304 5305-312,293

In one example, the guide RNA used in the present disclosure maycomprise at least one spacer sequence that, where “T” is “U”, may be anRNA sequence corresponding to a 20 nucleotide (nt) target sequence suchas, but not limited to, any of SEQ ID NO: 5305-312,293.

In one example, the guide RNA used in the present disclosure maycomprise at least one spacer sequence which, where “T” is “U,” is an RNAsequence corresponding to the 20 nt sequences such as, but not limitedto, any of SEQ ID NO: 5305-312,293.

In one example, a guide RNA may comprise a 20 nucleotide (nt) targetnucleic acid sequence associated with the PAM type such as, but notlimited to, NAAAAC, NNAGAAW, NNGRRT, NNNNGHTT, NRG, or YTN. As anon-limiting example, the 20 nt target nucleic acid sequence for aspecific target gene and a specific PAM type may be, where “T” is “U,”the RNA sequence corresponding to any one of the 20 nt nucleic acidsequences in Table 6.

TABLE 6 Nucleic Acid Sequences by PAM Type PAM: PAM: PAM: PAM: NAAAACNNAGAAW NNGRRT NNNNGHTT PAM: NRG PAM: YTN 20 nt Target 20 nt Target 20nt Target 20 nt Target 20 nt Target 20 nt Target Gene Nucleic AcidNucleic Acid Nucleic Acid Nucleic Acid Nucleic Acid Nucleic Acid SymbolSEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO ATXN15305-7241 7242-11,702 11,703-25,282 25,283-37,855  37,856- 155,356-155,355 312,293

In one example, a guide RNA may comprise a 20 nucleotide (nt) targetnucleic acid sequence associated with the YTN PAM type. As anon-limiting example, the 20 nt target nucleic acid sequence for aspecific target gene may comprise a 20 nt core sequence where the 20 ntcore sequence, where “T” is “U,” may be the RNA sequence correspondingto SEQ ID NO: 155,356-312,293. As another non-limiting example, the 20nt target nucleic acid sequence for a specific target gene may comprisea core sequence where the core sequence, where “T” is “U,” may be afragment, segment or region of the RNA sequence corresponding to any ofSEQ ID NO: 155,356-312,293.

VI. Other Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9.

CRISPR endonucleases, such as Cas9, can be used in the methods of thepresent disclosure. However, the teachings described herein, such astherapeutic target sites, could be applied to other forms ofendonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or usingcombinations of nucleases. However, in order to apply the teachings ofthe present disclosure to such endonucleases, one would need to, amongother things, engineer proteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease Fold. Because Fold functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active Fold dimer to form. Upondimerization of the Fold domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the Fold domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese Fold variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J BiolChem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

TALENs represent another format of modular nucleases whereby, as withZFNs, an engineered DNA binding domain is linked to the Fold nucleasedomain, and a pair of TALENs operate in tandem to achieve targeted DNAcleavage. The major difference from ZFNs is the nature of the DNAbinding domain and the associated target DNA sequence recognitionproperties. The TALEN DNA binding domain derives from TALE proteins,which were originally described in the plant bacterial pathogenXanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acidrepeats, with each repeat recognizing a single base pair in the targetDNA sequence that is typically up to 20 bp in length, giving a totaltarget sequence length of up to 40 bp. Nucleotide specificity of eachrepeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefitted fromthe use of obligate heterodimer variants of the Fold domain to reduceoff-target activity.

Additional variants of the Fold domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive Fold domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9 or CRISPR/Cpf1“nickase” mutants in which one of the Cas9 cleavage domains has beendeactivated. DNA nicks can be used to drive genome editing by HDR, butat lower efficiency than with a DSB. The main benefit is that off-targetnicks are quickly and accurately repaired, unlike the DSB, which isprone to NHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes. 39(12):e82 (2011); Li et al., Nucleic Acids Res.39(14):6315-25(2011); Weber et al., PLoS One. 6(2):e16765 (2011); Wanget al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); andCermak T et al., Methods Mol Biol. 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingLAGLIDADG (SEQ ID NO. 312,328), GIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK,and Vsr-like that are derived from a broad range of hosts, includingeukarya, protists, bacteria, archaea, cyanobacteria and phage. As withZFNs and TALENs, HEs can be used to create a DSB at a target locus asthe initial step in genome editing. In addition, some natural andengineered HEs cut only a single strand of DNA, thereby functioning assite-specific nickases. The large target sequence of HEs and thespecificity that they offer have made them attractive candidates tocreate site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012); and references cited therein.

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-TevI (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-FokI or dCpf1-Fok1 and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a Fold domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76(2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). BecauseFold must dimerize to become catalytically active, two guide RNAs arerequired to tether two Fold fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-TevI, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-TevI, with the expectation that off-targetcleavage can be further reduced.

VII. Kits

The present disclosure provides kits for carrying out the methodsdescribed herein. A kit can include one or more of a genome-targetingnucleic acid, a polynucleotide encoding a genome-targeting nucleic acid,a site-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the aspects of the methods described herein, or anycombination thereof.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a genome-targeting nucleic acid, (2) the site-directedpolypeptide or a vector comprising a nucleotide sequence encoding thesite-directed polypeptide, and (3) a reagent for reconstitution and/ordilution of the vector(s) and or polypeptide.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a genome-targeting nucleic acid, and (ii) a nucleotide sequenceencoding the site-directed polypeptide; and (2) a reagent forreconstitution and/or dilution of the vector.

In any of the above kits, the kit can comprise a single-molecule guidegenome-targeting nucleic acid. In any of the above kits, the kit cancomprise a double-molecule genome-targeting nucleic acid. In any of theabove kits, the kit can comprise two or more double-molecule guides orsingle-molecule guides. The kits can comprise a vector that encodes thenucleic acid targeting nucleic acid.

In any of the above kits, the kit can further comprise a polynucleotideto be inserted to effect the desired genetic modification.

Components of a kit can be in separate containers, or combined in asingle container.

Any kit described above can further comprise one or more additionalreagents, where such additional reagents are selected from a buffer, abuffer for introducing a polypeptide or polynucleotide into a cell, awash buffer, a control reagent, a control vector, a control RNApolynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, a diluting buffer, or thelike. A kit can also comprise one or more components that can be used tofacilitate or enhance the on-target binding or the cleavage of DNA bythe endonuclease, or improve the specificity of targeting.

In addition to the above-mentioned components, a kit can furthercomprise instructions for using the components of the kit to practicethe methods. The instructions for practicing the methods can be recordedon a suitable recording medium. For example, the instructions can beprinted on a substrate, such as paper or plastic, etc. The instructionscan be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or subpackaging), etc. The instructions can be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g. CD-ROM, diskette, flash drive, etc. In someinstances, the actual instructions are not present in the kit, but meansfor obtaining the instructions from a remote source (e.g. via theInternet), can be provided. An example of this case is a kit thatcomprises a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions can be recorded on a suitablesubstrate.

VIII. Methods, Compositions, and Therapeutics of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting methods according to the present disclosure:

In a first method, Method 1, the present disclosure provides a methodfor editing a ATXN1 gene in a cell by genome editing comprising:introducing into the cell one or more DNA endonucleases to effect one ormore SSBs or DSBs within or near the ATXN1 gene that results in apermanent deletion in the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the ATXN1 gene, thereby restoring the ATXN1 genefunction.

In another method, Method 2, the present disclosure provides a methodfor editing a ATXN1 gene in a cell by genome editing comprising:introducing into the cell one or more DNA endonucleases to effect one ormore SSBs or DSBs within or near the ATXN1 gene or ATXN1 regulatoryelements that results in one or more permanent insertion, deletion ormutation of at least one nucleotide within or near the ATXN1 gene and/ora permanent deletion in the expanded trinucleotide repeat, therebyreducing or eliminating the expression or function of aberrant ATXN1gene products.

In another method, Method 3, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder comprising: editing a patient specific iPSC within or near aATXN1 gene or other DNA sequences that encode regulatory elements of theATXN1 gene; differentiating the edited iPSC into a neuron or glial cellof the CNS; and implanting the neuron or glial cell of the CNS into thepatient.

In another method, Method 4, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 3, wherein the editing step comprises:introducing into the iPSC one or more DNA endonucleases to effect one ormore SSBs or DSBs within or near the ATXN1 gene that results in apermanent deletion of the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the ATXN1 gene and/or a permanent deletion in theexpanded trinucleotide repeat, thereby restoring the ATXN1 genefunction.

In another method, Method 5, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 3, wherein the editing step comprisesintroducing into the iPSC one or more DNA endonucleases to effect one ormore SSBs or DSBs within or near the ATXN1 gene or ATXN1 regulatoryelements that results in one or more permanent insertion, deletion ormutation of at least one nucleotide within or near the ATXN1 gene,thereby reducing or eliminating the expression or function of aberrantATXN1 gene products.

In another method, Method 6, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in any one of Methods 3-5, further comprising:creating the iPSC, wherein the creating step comprises: isolating asomatic cell from the patient; and introducing a set ofpluripotency-associated genes into the somatic cell to induce thesomatic cell to become the iPSC.

In another method, Method 7, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 6, wherein the somatic cell is afibroblast.

In another method, Method 8, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 6, wherein the set ofpluripotency-associated genes is one or more of the genes selected fromthe group consisting of: OCT4, SOX2, KLF4, Lin28, NANOG and cMYC.

In another method, Method 9, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder comprising: editing a mesenchymal stem cell within or near aATXN1 gene or other DNA sequences that encode regulatory elements of theATXN1 gene; differentiating the edited mesenchymal stem cell into aneuron or glial cell of the CNS; and implanting the neuron or glial cellof the CNS into the patient.

In another method, Method 10, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 9, wherein the editing step comprises:introducing into the mesenchymal stem cell one or more DNA endonucleasesto effect one or more SSBs or DSBs within or near the ATXN1 gene thatresults in a permanent deletion of the expanded trinucleotide repeat orreplacement of one or more nucleotide bases, or one or more exons and/orintrons within or near the ATXN1 gene, thereby restoring the ATXN1 genefunction.

In another method, Method 11, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 9, wherein the editing step comprises:introducing into the mesenchymal stem cell one or more DNA endonucleasesto effect one or more SSBs or DSBs within or near the ATXN1 gene orATXN1 regulatory elements that results in one or more permanentinsertion, deletion or mutation of at least one nucleotide within ornear the ATXN1 gene and/or a permanent deletion in the expandedtrinucleotide repeat, thereby reducing or eliminating the expression orfunction of aberrant ATXN1 gene products.

In another method, Method 12, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in any one of Methods 9-11, further comprising:isolating the mesenchymal stem cell from the patient, wherein themesenchymal stem cell is isolated from the patient's bone marrow orperipheral blood.

In another method, Method 13, the present disclosure provides an ex vivomethod for treating a patient having an ATXN1 related condition ordisorder, as provided in Method 12, wherein the isolating stepcomprises: aspiration of bone marrow and isolation of mesenchymal stemcells using density gradient centrifugation media.

In another method, Method 14, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder comprising: editing the ATXN1 gene in a cell of the patient.

In another method, Method 15, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder, as provided in Method 14, wherein the editing step comprises:introducing into the cell one or more DNA endonucleases to effect one ormore SSBs or DSBs within or near the ATXN1 gene that results in apermanent deletion of the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the ATXN1 gene, thereby restoring the ATXN1 genefunction.

In another method, Method 16, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder, as provided in Method 14, wherein the editing step comprises:introducing into the cell one or more DNA endonucleases to effect one ormore SSBs or DSBs within or near the ATXN1 gene or ATXN1 regulatoryelements that results in one or more permanent insertion, deletion ormutation of at least one nucleotide within or near the ATXN1 gene and/ora permanent deletion in the expanded trinucleotide repeat, therebyreducing or eliminating the expression or function of aberrant ATXN1gene products.

In another method, Method 17, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder, as provided in any one of Methods 14-16, wherein the cell is acell of the CNS.

In another method, Method 18, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder, as provided in Method 17 wherein the cell of the CNS is aneuron.

In another method, Method 19, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder, as provided in Method 17 wherein the cell of the CNS is aglial cell.

In another method, Method 20, the present disclosure provides an in vivomethod for treating a patient with an ATXN1 related condition ordisorder, as provided in any one of Methods 17-19, wherein the one ormore DNA endonuclease is delivered to the cell of the CNS via anyadministration route selected from the group consisting ofintraparenchymal, intravenous, intra-arterial, intracerebroventricular,intracisternal, intrathecal, intracranial or intraperitoneal routes.

In another method, Method 21, the present disclosure provides a methodfor treating a patient having an ATXN1 related condition or disorder, asprovided in any one of Methods 3, 9, or 14, wherein the ATXN1 relatedcondition or disorder is SCA1.

In another method, Method 22 the present disclosure provides a method ofaltering the contiguous genomic sequence of an ATXN1 gene in a cellcomprising: contacting the cell with one or more DNA endonuclease toeffect one or more SSBs or DSBs.

In another method, Method 23, the present disclosure provides a methodof altering the contiguous genomic sequence of an ATXN1 gene in a cell,as provided in Method 22, wherein the alteration of the contiguousgenomic sequence occurs in one or more exons of the ATXN1 gene.

In another method, Method 24, the present disclosure provides a methodof altering the contiguous genomic sequence of an ATXN1 gene in a cell,as provided in Method 23, wherein the alteration of the contiguousgenomic sequence occurs in exon 8 of the ATXN1 gene.

In another method, Method 25, the present disclosure provides a method,as provided in any one of Methods 1-24, wherein the one or more DNAendonuclease is selected from any of those sequences in SEQ ID NOs:1-620, and variants having at least 90% homology to any of thosesequences disclosed in SEQ ID NOs: 1-620.

In another method, Method 26, the present disclosure provides a method,as provided in Method 25, wherein the one or more DNA endonuclease isone or more protein or polypeptide.

In another method, Method 27, the present disclosure provides a method,as provided in Method 26, wherein the one or more protein or polypeptideis flanked at the N-terminus, the C-terminus, or both the N-terminus andC-terminus by one or more NLSs.

In another method, Method 28, the present disclosure provides a method,as provided in Method 27, wherein the one or more protein or polypeptideis flanked by two NLSs, one NLS located at the N-terminus and the secondNLS located at the C-terminus.

In another method, Method 29, the present disclosure provides a method,as provided in any one of Methods 27 or 28, wherein the one or more NLSsis a SV40 NLS.

In another method, Method 30, the present disclosure provides a method,as provided in Method 25, wherein the one or more DNA endonuclease isone or more polynucleotide encoding the one or more DNA endonuclease.

In another method, Method 31, the present disclosure provides a method,as provided in Method 30, wherein the one or more DNA endonuclease isone or more RNA encoding the one or more DNA endonuclease.

In another method, Method 32, the present disclosure provides a method,as provided in Method 31, wherein the one or more RNA is one or morechemically modified RNA.

In another method, Method 33, the present disclosure provides a method,as provided in Method 32, wherein the one or more RNA is chemicallymodified in the coding region.

In another method, Method 34, the present disclosure provides a method,as provided in any one of Methods 30-33, wherein the one or morepolynucleotide or one or more RNA is codon optimized.

In another method, Method 35, the present disclosure provides a method,as provided in any one of Methods 1-34, wherein the method furthercomprises introducing one or more gRNA or one or more sgRNA.

In another method, Method 36, the present disclosure provides a method,as provided in Method 35, wherein the one or more gRNA or one or moresgRNA comprises a spacer sequence that is complementary to a sequencewithin or near the expanded trinucleotide repeat in the ATXN1 gene.

In another method, Method 37, the present disclosure provides a method,as provided in Method 35, wherein the one or more gRNA or one or moresgRNA comprises a spacer sequence that is complementary to a DNAsequence within or near the ATXN1 gene.

In another method, Method 38, the present disclosure provides a method,as provided in Method 35, wherein the one or more gRNA or one or moresgRNA comprises a spacer sequence that is complementary to a sequenceflanking the ATXN1 gene or other sequence that encodes a regulatoryelement of the ATXN1 gene.

In another method, Method 39, the present disclosure provides a method,as provided in Method 35, wherein the one or more gRNA or one or moresgRNA comprises a spacer sequence that is complementary to a DNAsequence within or near exon 8 of the ATXN1 gene.

In another method, Method 40, the present disclosure provides a method,as provided in any one of Methods 35-39, wherein the one or more gRNA orone or more sgRNA is chemically modified.

In another method, Method 41, the present disclosure provides a method,as provided in Method 40, wherein the one or more modified sgRNAscomprises three 2′-O-methyl-phosphorothioate residues at or near each ofits 5′ and 3′ ends.

In another method, Method 42, the present disclosure provides a method,as provided in any one of Methods 39-44, wherein the one or more gRNA orone or more sgRNA comprises a RNA sequence corresponding to a sequenceselected from the group consisting of SEQ ID NOs: 42685, 42504, 101247,42473, 101253, 101251, 101250, 101085, 101252, 101259, 101139, 101314,42644, 42421, 101103, 42634, 101054, 101319, 101044, 101152, 101320,42637, 101060, 101150, 101062, 42605, 101249, 101316, 101061, 101248,101296, 42686, 101093, 101137, 101236, 101153, 101313, 42451, 101045,42457, 42416, 42474, 42458, 42450, 101117, 42566, 42417, 101151, 42475,101174, 101106, 101069, 101295, 101084, 42449, 101140, 101261, 101196,101136, 101104, 101177, 101308, 42446, 42447, 42635, 101068, 101176, and101064.

In another method, Method 43, the present disclosure provides a method,as provided in any one of Methods 35-40, wherein the one or more gRNA orone or more sgRNA is pre-complexed with the one or more DNA endonucleaseto form one or more ribonucleoproteins (RNPs).

In another method, Method 44, the present disclosure provides a method,as provided in Method 43, wherein the pre-complexing involves a covalentattachment of the one or more gRNA or one or more sgRNA to the one ormore DNA endonuclease.

In another method, Method 45, the present disclosure provides a method,as provided in any one of Methods 43 or 44, wherein the weight ratio ofsgRNA to DNA endonuclease in the RNP is 1:1.

In another method, Method 46, the present disclosure provides a method,as provided in any one of Methods 25-45, wherein the one or more DNAendonuclease is formulated in a liposome or lipid nanoparticle.

In another method, Method 47, the present disclosure provides a method,as provided in any one of Methods 35-45, wherein the one or more DNAendonuclease is formulated in a liposome or a lipid nanoparticle whichalso comprises the one or more gRNA or one or more sgRNA.

In another method, Method 48, the present disclosure provides a method,as provided in any one of Methods 25 or 35-42, wherein the one or moreDNA endonuclease is encoded in an AAV vector particle.

In another method, Method 49, the present disclosure provides a method,as provided in any one of Methods 35-42, wherein the one or more gRNA orone or more sgRNA is encoded in an AAV vector particle.

In another method, Method 50, the present disclosure provides a method,as provided in any one of Methods 35-42, wherein the one or more DNAendonuclease is encoded in an AAV vector particle which also encodes theone or more gRNA or one or more sgRNA.

In another method, Method 51, the present disclosure provides a method,as provided in any one of Methods 48-50, wherein the AAV vector particleis selected from the group consisting of any of those sequencesdisclosed in SEQ ID NOs: 4734-5302 and Table 2.

In another method, Method 52, the present disclosure provides a method,as provided in any one of Methods 1-51, wherein the method furthercomprises introducing into the cell a donor template comprising at leasta portion of the wild-type ATXN1 gene.

In another method, Method 53, the present disclosure provides a method,as provided in Method 52, wherein the at least a portion of thewild-type ATXN1 gene comprises one or more sequences selected from thegroup consisting of an ATXN1 exon, an ATXN1 intron, and a sequencecomprising an exon:intron junction of ATXN1.

In another method, Method 54, the present disclosure provides a method,as provided in any one of Methods 52 or 53, wherein the donor templatecomprises homologous arms to the genomic locus of the ATXN1 gene.

In another method, Method 55, the present disclosure provides a method,as provided in any one of Methods 52-54, wherein the donor template iseither a single or double stranded polynucleotide.

In another method, Method 56, the present disclosure provides a method,as provided in any one of Methods 52-55, wherein the donor template isencoded in an AAV vector particle, where the AAV vector particle isselected from the group consisting of any of those disclosed in SEQ IDNOs: 4734-5302 and Table 2.

In another method, Method 57, the present disclosure provides a method,as provided in any one of Methods 52-55, wherein the one or morepolynucleotide encoding one or more DNA endonuclease is formulated intoa lipid nanoparticle, and the one or more gRNA or one or more sgRNA isdelivered to the cell ex vivo by electroporation and the donor templateis delivered to the cell by an AAV vector.

In another method, Method 58, the present disclosure provides a method,as provided in any one of Methods 52-55, wherein the one or morepolynucleotide encoding one or more DNA endonuclease is formulated intoa liposome or lipid nanoparticle which also comprises the one or moregRNA or one or more sgRNA and the donor template.

In another method, Method 59, the present disclosure provides a method,as provided in any one of Methods 1-58, wherein the ATXN1 gene islocated on Chromosome 6: 16,299,112-16,761,491 (Genome ReferenceConsortium—GRCh38).

In a first composition, Composition 1, the present disclosure provides asingle-molecule guide RNA comprising at least a spacer sequence that isan RNA sequence corresponding to any one of SEQ ID NOs: 5305-312,293.

In another composition, Composition 2, the present disclosure providesthe single-molecule guide RNA of Composition 1, wherein thesingle-molecule guide RNA further comprises a spacer extension region.

In another composition, Composition 3, the present disclosure providesthe single-molecule guide RNA of Composition 1, wherein thesingle-molecule guide RNA further comprises a tracrRNA extension region.

In another composition, Composition 4, the present disclosure providesthe single-molecule guide RNA of any one of Compositions 1-3, whereinthe single-molecule guide RNA is chemically modified.

In another composition, Composition 5, the present disclosure providesthe single-molecule guide RNA of any one of Compositions 1-4, whereinthe single-molecule guide RNA is pre-complexed with a DNA endonuclease.

In another composition, Composition 6, the present disclosure providesthe single-molecule guide RNA of Composition 5, wherein the DNAendonuclease is a Cas9 or Cpf1 endonuclease.

In another composition, Composition 7, the present disclosure providesthe single-molecule guide RNA of Composition 6, wherein the Cas9 or Cpf1endonuclease is selected from the group consisting of S. pyogenes Cas9,S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR1 Cas9, S.thermophilus CRISPR 3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpf1and Acidaminococcus sp. BV3L6 Cpf1, and variants having at least 90%homology to the endonucleases.

In another composition, Composition 8, the present disclosure providesthe single-molecule guide RNA of Composition 7, wherein the Cas9 or Cpf1endonuclease comprises one or more NLSs.

In another composition, Composition 9, the present disclosure providesthe single-molecule guide RNA of Composition 8, wherein at least one NLSis at or within 50 amino acids of the amino-terminus of the Cas9 or Cpf1endonuclease and/or at least one NLS is at or within 50 amino acids ofthe carboxy-terminus of the Cas9 or Cpf1 endonuclease.

In another composition, Composition 10, the present disclosure providesa DNA encoding the single-molecule guide RNA of any one of Compositions1-4.

In a first therapeutic, Therapeutic 1, the present disclosure provides atherapeutic comprising at least one or more gRNAs for editing an ATXN1gene in a cell from a patient with an ATXN1 related condition ordisorder, the one or more gRNAs comprising a spacer sequence selectedfrom the group consisting of nucleic acid sequences in any one of SEQ IDNOs: 5305-312,293 of the Sequence Listing.

In another therapeutic, Therapeutic 2, the present disclosure provides atherapeutic for treating a patient with an ATXN1 related condition ordisorder formed by the method comprising: introducing one or more DNAendonucleases; introducing one or more gRNA or one or more sgRNA forediting a ATXN1 gene; wherein the one or more gRNAs or sgRNAs comprise aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5305-312,293 of the Sequence Listing.

In another therapeutic, Therapeutic 3, the present disclosure providesthe therapeutic of any one of Therapeutics 1 or 2, wherein the ATXN1related condition or disorder is SCA1.

IX. Definitions

In addition to the definitions previously set forth herein, thefollowing definitions are relevant to the present disclosure:

The term “comprising” or “comprises” is used in reference tocompositions, methods, therapeutics, and respective component(s)thereof, that are essential to the present disclosure, yet open to theinclusion of unspecified elements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given aspect. The term permits the presence of additional elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that aspect of the present disclosure.

The term “consisting of” refers to compositions, methods, therapeuticsand respective components thereof as described herein, which areexclusive of any element not recited in that description of the aspect.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Any numerical range recited in this specification describes allsub-ranges of the same numerical precision (i.e., having the same numberof specified digits) subsumed within the recited range. For example, arecited range of “1.0 to 10.0” describes all sub-ranges between (andincluding) the recited minimum value of 1.0 and the recited maximumvalue of 10.0, such as, for example, “2.4 to 7.6,” even if the range of“2.4 to 7.6” is not expressly recited in the text of the specification.Accordingly, the Applicant reserves the right to amend thisspecification, including the claims, to expressly recite any sub-rangeof the same numerical precision subsumed within the ranges expresslyrecited in this specification. All such ranges are inherently describedin this specification such that amending to expressly recite any suchsub-ranges will comply with written description, sufficiency ofdescription, and added matter requirements, including the requirementsunder 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expresslyspecified or otherwise required by context, all numerical parametersdescribed in this specification (such as those expressing values,ranges, amounts, percentages, and the like) may be read as if prefacedby the word “about,” even if the word “about” does not expressly appearbefore a number. Additionally, numerical parameters described in thisspecification should be construed in light of the number of reportedsignificant digits, numerical precision, and by applying ordinaryrounding techniques. It is also understood that numerical parametersdescribed in this specification will necessarily possess the inherentvariability characteristic of the underlying measurement techniques usedto determine the numerical value of the parameter.

The details of one or more aspects of the present disclosure are setforth in the accompanying description below. Although any materials andmethods similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferredmaterials and methods are now described. Other features, objects andadvantages of the present disclosure will be apparent from thedescription. In the description, the singular forms also include theplural unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this present disclosure belongs. In the case of conflict, thepresent description will control.

The present disclosure is further illustrated by the followingnon-limiting examples.

X. Examples

The present disclosure will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting aspects ofthe present disclosure.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicdeletions, insertions, or replacements, termed “genomic modifications”herein, in the ATXN1 gene that lead to permanent deletion or correctionof expanded trinucleotide repeats in the ATXN1 gene, knock-out of theATXN1 gene, or correction of the entire gene, or correction of mutationswithin the gene, that restore the wild-type or similar levels oftrinucleotide repeats in the ATXN1 gene and/or eliminate aberrant ATXN1gene products.

All tested gRNAs can be used for an HDR/correction based editingapproach. Selected pairs of gRNAs can be used to make deletions in theATXN1 gene that restore the reading frame. Selected pairs of gRNAs canbe used to make mutations that simulate patient mutations and can beused to generate model ATXN1 mutant lines.

Various Cas orthologs are evaluated for cutting. gRNAs are delivered asRNA and expressed from the U6 promoter in plasmids. The correspondingCas protein is either knocked into the cell line of interest andconstitutively expressed, delivered as mRNA, or delivered as protein.The gRNA activity in all formats is evaluated using TIDE analysis inHEK293T cells.

Introduction of the defined therapeutic modifications described aboverepresents a novel therapeutic strategy for the potential ameliorationof Spinocerebellar ataxia 1 (SCA1) and related disorders, as describedand illustrated herein.

Example 1—CRISPR/S. pyogenes(Sp)Cas9 Target Sites for the ATXN1 Gene

Regions of the ATXN1 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 37,856-155,355 of the SequenceListing.

Example 2—CRISPR/S. aureus(Sa)Cas9 Target Sites for the ATXN1 Gene

Regions of the ATXN1 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 11,703-25,282 of the SequenceListing.

Example 3—CRISPR/S. thermophilus(St)Cas9 Target Sites for the ATXN1 Gene

Regions of the ATXN1 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 7242-11,702 of the Sequence Listing.

Example 4—CRISPR/T. denticola(Td)Cas9 Target Sites for the ATXN1 Gene

Regions of the ATXN1 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 5305-7241 of the Sequence Listing.

Example 5—CRISPR/N. meningitidis(Nm)Cas9 Target Sites for the ATXN1 Gene

Regions of the ATXN1 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNNNGHTT. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 25,283-37,855 of the SequenceListing.

Example 6—CRISPR/Cpf1 Target Sites for the ATXN1 Gene

Regions of the ATXN1 gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence TTNor YTN. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 155,356-312,293 of the SequenceListing.

Example 7—Bioinformatics Analysis of the Guide Strands

Candidate guides were then screened and selected in a single process ormulti-step process that involves both theoretical binding andexperimentally assessed activity at both on-target and off-target sites.By way of illustration, candidate guides having sequences that match aparticular on-target site, such as a site within the ATXN1 gene, withadjacent PAM can be assessed for their potential to cleave at off-targetsites having similar sequences, using one or more of a variety ofbioinformatics tools available for assessing off-target binding, asdescribed and illustrated in more detail below, in order to assess thelikelihood of effects at chromosomal positions other than thoseintended.

Candidates predicted to have relatively lower potential for off-targetactivity can then be assessed experimentally to measure their on-targetactivity, and then off-target activities at various sites. Preferredguides have sufficiently high on-target activity to achieve desiredlevels of gene editing at the selected locus, and relatively loweroff-target activity to reduce the likelihood of alterations at otherchromosomal loci. The ratio of on-target to off-target activity is oftenreferred to as the “specificity” of a guide.

For initial screening of predicted off-target activities, there are anumber of bioinformatics tools known and publicly available that can beused to predict the most likely off-target sites; and since binding totarget sites in the CRISPR/Cas9 or CRISPR/Cpf1 nuclease system is drivenby Watson-Crick base pairing between complementary sequences, the degreeof dissimilarity (and therefore reduced potential for off-targetbinding) is essentially related to primary sequence differences:mismatches and bulges, i.e. bases that are changed to anon-complementary base, and insertions or deletions of bases in thepotential off-target site relative to the target site. An exemplarybioinformatics tool called COSMID (CRISPR Off-target Sites withMismatches, Insertions and Deletions) (available on the web atcrispr.bme.gatech.edu) compiles such similarities. Other bioinformaticstools include, but are not limited to autoCOSMID and CCtop.

Bioinformatics were used to minimize off-target cleavage in order toreduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof off-target activity due to non-specific hybridization of the guidestrand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it isimportant to have a bioinformatics tool that can identify potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.Bioinformatics tools based upon the off-target algorithm CCTop were usedto search genomes for potential CRISPR off-target sites (CCTop isavailable on the web at crispr.cos.uni-heidelberg.de/). The outputranked lists of the potential off-target sites based on the number andlocation of mismatches, allowing more informed choice of target sites,and avoiding the use of sites with more likely off-target cleavage.

Additional bioinformatics pipelines were employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that may be used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors areweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

Initial evaluation and screening of CRISPR/Cas9 target sites focusedwithin exon 8, the region upstream of exon 8, and the region downstreamof exon 8. gRNAs targeting exon 8 can be used to insert full length cDNAof ATXN1 for replacing the mutated endogenous ATXN1 gene and restoringprotein expression. Guide RNAs targeting exon 8 can be used to targetthe expanded repeats. Pairs of gRNAs on either side of the expandedrepeat sequence can be used to excise the expanded repeats. gRNAsoverlapping with expanded repeat sequence can be used to induce partialor full contraction of the repeats. In addition, carefully chosen gRNAstogether with dCas9 fused to chromatin modifier proteins can be used toalleviate the epigenetic changes that occur at the ATXN1 allele withexpanded repeats and restore ATXN1 expression. These gRNAs can also beused to edit one or more nucleotides, exonic sequences and/or intronicsequences.

Initial bioinformatics analysis identified, 605 gRNAs targeting withinexon 8, the region upstream of exon 8, and the region downstream of exon8. Further analysis of predicted off-target sites and evaluation of thegRNA target site within the ATXN1 gene resulted in 68 gRNAs selected forscreening. The prioritized list of 68 single gRNAs targeting the ATXN1gene were tested for cutting efficiencies using SpCas9 (Table 7).

TABLE 7 SEQ ID NO. Name Sequence 42685 SCA1_T67 GGGAGTTCACACCGAGTCCA42504 SCA1_T3 GTCATGCAATACGCCGACTC 101247 SCA1_T52 CGGCAGGTGAGCGTACTGCA42473 SCA1_T23 GGAGAAGAGCCGGCGGTACG 101253 SCA1_T79 GTACTGCACGGGGGACACCG101251 SCA1_T91 GCGTACTGCACGGGGGACAC 101250 SCA1_T11CAGGTGAGCGTACTGCACGG 101085 SCA1_T44 GCTGCGGGCGTGTTGCTGTT 101252SCA1_T83 CGTACTGCACGGGGGACACC 101259 SCA1_T45 TGGCTGCGGGGTGGCGTACG101139 SCA1_T61 GTATTGCATGACGACCTGGG 101314 SCA1_T18GCGGGGATCTCGCGCTTCTT 42644 SCA1_T115 CGCCACCAGCCGGTCCTCCG 42421 SCA1_T87GCTTCAGAGCCACTCCCGGT 101103 SCA1_T32 ACCACGTGCCTGGACTCGTA 42634 SCA1_T97AGCGACAACCACCGGGTGGA 101054 SCA1_T105 GTGTGGTCTGAATGACCGTG 101319SCA1_T55 GGCATTCGTTGCTCCGCTCT 101044 SCA1_T36 GGGTTGAGTCCCTGCGTAGA101152 SCA1_T48 GTGTGGGATCATCGTCTGGT 101320 SCA1_T59TTCGTTGCTCCGCTCTTGGT 42637 SCA1_T71 GCCCAGCGACAACCACCGGG 101060 SCA1_T38CGTGTGGGGTGAGAGCGCGT 101150 SCA1_T12 GCGTGTGTGGGATCATCGTC 101062SCA1_T50 GTGAGAGCGCGTAGGACCGG 42605 SCA1_T84 GGTGGAGCTTGGTTTACAAC 101249SCA1_T13 GCAGGTGAGCGTACTGCACG 101316 SCA1_T100 GGGATCTCGCGCTTCTTGGG101061 SCA1_T41 GGGGTGAGAGCGCGTAGGAC 101248 SCA1_T43GGCAGGTGAGCGTACTGCAC 101296 SCA1_T19 TCCACCCGGTGGTTGTCGCT 42686 SCA1_T73TGGGAGTTCACACCGAGTCC 101093 SCA1_T102 ACAGAGGCCCGGACCCCCGA 101137SCA1_T16 GGCGTATTGCATGACGACCT 101236 SCA1_T98 TGGCATAGGTTCCACTGTAT101153 SCA1_T110 TGTGGGATCATCGTCTGGTG 101313 SCA1_T22GGCGGGGATCTCGCGCTTCT 42451 SCA1_T64 CTACAGCAGTCGTGATCCTT 101045 SCA1_T31AGTCCCTGCGTAGAAGGCCG 42457 SCA1_T29 CCCGTACGAGTCCAGGCACG 42416 SCA1_T72CAGCCACGGCCTTCTACGCA 42474 SCA1_T26 TGGAGAAGAGCCGGCGGTAC 42458 SCA1_T8TTCCTCACCCGTACGAGTCC 42450 SCA1_T51 TACAGCAGTCGTGATCCTTC 101117 SCA1_T14AGGACGGGGCCCCGTACCGC 42566 SCA1_T113 GGAGGCCTATTCCACTCTGC 42417 SCA1_T37CCAGCCACGGCCTTCTACGC 101151 SCA1_T65 TGTGTGGGATCATCGTCTGG 42475 SCA1_T34ATGGAGAAGAGCCGGCGGTA 101174 SCA1_T75 GCCGGTGTTCTGCGGAGAAC 101106SCA1_T10 TGCCTGGACTCGTACGGGTG 101069 SCA1_T92 CAGGCCACTTTTGTCGTTGA101295 SCA1_T4 CTCCACCCGGTGGTTGTCGC 101084 SCA1_T112AGCTGCGGGCGTGTTGCTGT 42449 SCA1_T107 ACAGCAGTCGTGATCCTTCG 101140SCA1_T103 TATTGCATGACGACCTGGGA 101261 SCA1_T40 TGCGGGGTGGCGTACGCGGC101196 SCA1_T60 CAGCCTTGTGTCCCGGCGTC 101136 SCA1_T7 CGGCGTATTGCATGACGACC101104 SCA1_T35 CCACGTGCCTGGACTCGTAC 101177 SCA1_T80TGTGGACGTACTGGTTCTGC 101308 SCA1_T63 CTTCTCCTCGGAGGACCGGC 42446 SCA1_T78TCGTGATCCTTCGGGGGTCC 42447 SCA1_T30 GTCGTGATCCTTCGGGGGTC 42635 SCA1_T86CAGCGACAACCACCGGGTGG 101068 SCA1_T58 GCAGGCCACTTTTGTCGTTG 101176SCA1_T90 AACTGGAAATGTGGACGTAC 101064 SCA1_T47 AGCGCGTAGGACCGGTGGCC

Note that the SEQ ID NOs represent the DNA sequence of the genomictarget, while the gRNA or sgRNA spacer sequence will be the RNA versionof the DNA sequence.

Example 8—Testing of Preferred Guides in In Vitro Transcribed (IVT) gRNAScreen

To identify a large spectrum of pairs of gRNAs able to edit the cognateDNA target region, an in vitro transcribed (IVT) gRNA screen wasconducted. The relevant genomic sequence was submitted for analysisusing a gRNA design software. The resulting list of gRNAs was narrowedto a select list of gRNAs as described above based on uniqueness ofsequence (only gRNAs without a perfect match somewhere else in thegenome were screened) and minimal predicted off targets. This set ofgRNAs was in vitro transcribed, and transfected using LipofectamineMessengerMAX into HEK293T cells that constitutively express Cas9. Cellswere harvested 48 hours post transfection, the genomic DNA was isolated,and cutting efficiency was evaluated using TIDE analysis. (FIGS. 2-3).

The gRNA or pairs of gRNA with significant activity can then be followedup in cultured cells to measure correction of the ATXN1 mutation.Off-target events can be followed again. A variety of cells can betransfected and the level of gene correction and possible off-targetevents measured. These experiments allow optimization of nuclease anddonor design and delivery.

Example 9—Testing of Preferred Guides in Cells for Off-Target Activity

The gRNAs having the best on-target activity from the IVT screen in theabove example are tested for off-target activity using Hybrid captureassays, GUIDE Seq. and whole genome sequencing in addition to othermethods.

Example 10—Testing Different Approaches for HDR Gene Editing

After testing the gRNAs for both on-target activity and off-targetactivity, repeat expansion correction and whole gene correction strategy(together with disruption of the mutant gene) will be tested for HDRgene editing.

For the whole gene correction approach, a single-stranded ordouble-stranded DNA having homologous arms to the ATXN1 chromosomalregion may include more than 40 nt of the first exon (the first codingexon or upstream sequence) of the ATXN1 gene, the complete CDS of theATXN1 gene and 3′ UTR of the ATXN1 gene, and at least 40 nt of thefollowing intron. The single-stranded or double-stranded DNA havinghomologous arms to the ATXN1 chromosomal region may include more than 80nt of the first exon of the ATXN1 gene, the complete CDS of the ATXN1gene and 3′ UTR of the ATXN1 gene, and at least 80 nt of the followingintron. The single-stranded or double-stranded DNA having homologousarms to the ATXN1 chromosomal region may include more than 100 nt of thefirst exon of the ATXN1 gene, the complete CDS of the ATXN1 gene and 3′UTR of the ATXN1 gene, and at least 100 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to theATXN1 chromosomal region may include more than 150 nt of the first exonof the ATXN1 gene, the complete CDS of the ATXN1 gene and 3′ UTR of theATXN1 gene, and at least 150 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to theATXN1 chromosomal region may include more than 300 nt of the first exonof the ATXN1 gene, the complete CDS of the ATXN1 gene and 3′ UTR of theATXN1 gene, and at least 300 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to theATXN1 chromosomal region may include more than 400 nt of the first exonof the ATXN1 gene, the complete CDS of the ATXN1 gene and 3′ UTR of theATXN1 gene, and at least 400 nt of the following intron.

Alternatively, the DNA template will be delivered by a recombinant AAVparticle, or other delivery vectors, such as those taught herein.

A knock-in of ATXN1 cDNA can be performed into any selected chromosomallocation, including the ATXN1 gene locus or in a safe harbor locus,e.g.: AAVS1 (intron 1 of PPP1R12C), HPRT, H11, hRosa26, and/or F-Aregion. Assessment of efficiency of HDR mediated knock-in of cDNA intothe first exon can utilize cDNA knock-in into safe harbor loci such as:single-stranded or double-stranded DNA having homologous arms to one ofthe following regions, for example: exon 1, intron 1 or exon 2 ofPPP1R12C; exon 1, intron 1 or exon 2 of HPRT; and/or exon 1, intron 1 orexon 2 of hRosa26; 5′UTR correspondent to ATXN1 or alternative 5′ UTR,complete CDS of ATXN1 and 3′ UTR of ATXN1 or modified 3′ UTR and atleast 80 nt of the first intron, alternatively same DNA templatesequence will be delivered by AAV, or other viral (or non-viral)delivery vector.

Example 11—Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

After testing the different strategies for gene editing, the leadCRISPR-Cas9/DNA donor combinations will be re-assessed in cells forefficiency of deletion, recombination, and off-target specificity. Cas9mRNA or RNP will be formulated into lipid nanoparticles for delivery,sgRNAs will be formulated into nanoparticles or delivered as arecombinant AAV particle, and donor DNA will be formulated intonanoparticles or delivered as recombinant AAV particle.

Example 12—In Vivo Testing in Relevant Animal Model

After the CRISPR-Cas9/DNA donor combinations have been re-assessed, thelead formulations will be tested in vivo in an animal model.

Culture in human cells allows direct testing on the human target and thebackground human genome, as described above.

Preclinical efficacy and safety evaluations can be observed throughengraftment of modified mouse or human neurons in a mouse model. Themodified cells can be observed in the months after engraftment.

XI. Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificexamples in accordance with the invention described herein. The scope ofthe present disclosure is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The present disclosure includes examples inwhich exactly one member of the group is present in, employed in, orotherwise relevant to a given product or process. The present disclosureincludes examples in which more than one, or the entire group membersare present in, employed in, or otherwise relevant to a given product orprocess.

In addition, it is to be understood that any particular example of thepresent disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such examples aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular example of the compositions of the present disclosure (e.g.,any antibiotic, therapeutic or active ingredient; any method ofproduction; any method of use; etc.) can be excluded from any one ormore claims, for any reason, whether or not related to the existence ofprior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the present disclosure in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described examples, it isnot intended that it should be limited to any such particulars orexamples or any particular example, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

NOTE REGARDING ILLUSTRATIVE EXAMPLES

While the present disclosure provides descriptions of various specificaspects for the purpose of illustrating various aspects of the presentdisclosure and/or its potential applications, it is understood thatvariations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative aspects provided herein.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

1. A method for editing a Spinocerebellar Ataxia Type 1 Protein (ATXN1)gene in a cell by genome editing comprising: introducing into the cellone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the ATXN1 gene that results in a permanent deletion in theexpanded trinucleotide repeat or replacement of one or more nucleotidebases, or one or more exons and/or introns within or near the ATXN1gene, thereby restoring the ATXN1 gene function.
 2. (canceled)
 3. An exvivo method for treating a patient having an ATXN1 related condition ordisorder comprising: (a) editing a patient specific induced pluripotentstem cell (iPSC) or a mesenchymal stem cell according to the method ofclaim 1; (b) differentiating the edited cell of (a) into a neuron orglial cell of the Central Nervous System (CNS); and (c) implanting theneuron or glial cell of the Central Nervous System (CNS) into thepatient. 4.-13. (canceled)
 14. An in vivo method for treating a patientwith an ATXN1 related condition or disorder comprising: editing theATXN1 gene in a cell of the patient according to the method of claim 1,optionally wherein the cell is a cell of the Central Nervous System(CNS) such as a neuron or a glial cell. 15.-24. (canceled)
 25. Themethod of claim 1, wherein the one or more DNA endonuclease is selectedfrom any of those sequences disclosed in SEQ ID NOs: 1-620, or variantshaving at least 90% homology to any of those sequences disclosed in SEQID NOs: 1-620.
 26. The method of claim 25, wherein the one or more DNAendonuclease is provided as: one or more protein or polypeptide; or oneor more polynucleotide encoding the one or more DNA endonuclease,wherein the one or more polynucleotide is codon optimized, and when theone or more polynucleotide is one or more ribonucleic acid (RNA), theone or more RNA is optionally chemically modified, optionally in thecoding region.
 27. The method of claim 25, wherein the one or more DNAendonuclease is flanked at the N-terminus, the C-terminus, or both theN-terminus and C-terminus by one or more nuclear localization signals(NLSs). 28.-34. (canceled)
 35. The method of claim 25, wherein themethod further comprises introducing one or more gRNA or one or moresgRNA, wherein the one or more gRNA or one or more sgRNA comprises aspacer sequence that is complementary to: a sequence within or near theexpanded trinucleotide repeat in the ATXN1 gene; a sequence within ornear the ATXN1 gene; a sequence flanking the ATXN1 gene or othersequence that encodes a regulatory element of the ATXN1 gene; or asequence within or near exon 8 of the ATXN1 gene. 36.-39. (canceled) 40.The method of claim 35, wherein the one or more gRNA or one or moresgRNA: is chemically modified; comprises three2′-O-methyl-phosphorothioate residues at or near each of its 5′ and 3′ends; is pre-complexed with the one or more DNA endonuclease to form oneor more ribonucleoproteins (RNPs); or a combination thereof. 41.(canceled)
 42. The method of claim 35, wherein the one or more gRNA orone or more sgRNA comprises a RNA sequence corresponding to a sequenceselected from the group consisting of SEQ ID NOs: 42685, 42504, 101247,42473, 101253, 101251, 101250, 101085, 101252, 101259, 101139, 101314,42644, 42421, 101103, 42634, 101054, 101319, 101044, 101152, 101320,42637, 101060, 101150, 101062, 42605, 101249, 101316, 101061, 101248,101296, 42686, 101093, 101137, 101236, 101153, 101313, 42451, 101045,42457, 42416, 42474, 42458, 42450, 101117, 42566, 42417, 101151, 42475,101174, 101106, 101069, 101295, 101084, 42449, 101140, 101261, 101196,101136, 101104, 101177, 101308, 42446, 42447, 42635, 101068, 101176, and101064. 43.-45. (canceled)
 46. The method of claim 35, wherein: the oneor more DNA endonuclease is formulated in a liposome or lipidnanoparticle; the one or more DNA endonuclease is formulated in aliposome or a lipid nanoparticle which also comprises the one or moregRNA or one or more sgRNA; the one or more DNA endonuclease is encodedin an AAV vector particle; the one or more gRNA or one or more sgRNA isencoded in an AAV vector particle; or wherein the one or more DNAendonuclease is encoded in an AAV vector particle which also encodes theone or more gRNA or one or more sgRNA. 47.-51. (canceled)
 52. The methodof claim 26, wherein the method further comprises introducing into thecell a donor template comprising at least a portion of the wild-typeATXN1 gene, wherein the donor template: comprises homologous arms to thegenomic locus of the ATXN1 gene; is a single or double strandedpolynucleotide; is encoded in an AAV vector particle, or a combinationthereof.
 53. The method of claim 52, wherein the at least a portion ofthe wild-type ATXN1 gene comprises one or more sequences selected fromthe group consisting of an ATXN1 exon, an ATXN1 intron, and a sequencecomprising an exon:intron junction of ATXN1. 54.-56. (canceled)
 57. Themethod of claim 52, wherein the one or more polynucleotide encoding oneor more DNA endonuclease is formulated into a lipid nanoparticle, andthe one or more gRNA or one or more sgRNA is delivered to the cell exvivo by electroporation and the donor template is delivered to the cellby an adeno-associated virus (AAV) vector, or the one or morepolynucleotide encoding one or more DNA endonuclease is formulated intoa liposome or lipid nanoparticle which also comprises the one or moregRNA or one or more sgRNA and the donor template. 58.-59. (canceled) 60.A single-molecule guide RNA comprising at least a spacer sequence thatis an RNA sequence corresponding to any one of SEQ ID NOs: 5305-312,293.61. The single-molecule guide RNA of claim 60, wherein thesingle-molecule guide RNA: further comprises a spacer extension region;further comprises a tracrRNA extension region; is chemically modified;or a combination thereof. 62.-63. (canceled)
 64. The single-moleculeguide RNA of claim 61 pre-complexed with a DNA endonuclease, wherein theDNA endonuclease is a Cas9 or Cpf1 endonuclease.
 65. (canceled)
 66. Thesingle-molecule guide RNA of claim 64, wherein the Cas9 or Cpf1endonuclease is selected from the group consisting of S. pyogenes Cas9,S. aureus Cas9, N. meningitidis Cas9, S. thermophilus CRISPR1 Cas9, S.thermophilus CRISPR 3 Cas9, T denticola Cas9, L. bacterium ND2006 Cpf1and Acidaminococcus sp. BV3L6 Cpf1, and variants having at least 90%homology to the endonucleases.
 67. The single-molecule guide RNA ofclaim 66, wherein the Cas9 or Cpf1 endonuclease comprises one or morenuclear localization signals (NLSs), wherein the at least one NLS is ator within 50 amino acids of the amino-terminus of the Cas9 or Cpf1endonuclease and/or at least one NLS is at or within 50 amino acids ofthe carboxy-terminus of the Cas9 or Cpf1 endonuclease.
 68. (canceled)69. A DNA encoding the single-molecule guide RNA of claim
 60. 70.(canceled)
 71. A therapeutic for treating a patient with an ATXN1related condition or disorder formed by the method comprising:introducing one or more DNA endonucleases; introducing one or more gRNAor one or more sgRNA for editing a ATXN1 gene; wherein the one or moregRNAs or sgRNAs comprise a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5305-312,293 of theSequence Listing.
 72. (canceled)